Sample records for quantitative proteomics approaches
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Standardization approaches in absolute quantitative proteomics with mass spectrometry.
Calderón-Celis, Francisco; Encinar, Jorge Ruiz; Sanz-Medel, Alfredo
2017-07-31
Mass spectrometry-based approaches have enabled important breakthroughs in quantitative proteomics in the last decades. This development is reflected in the better quantitative assessment of protein levels as well as to understand post-translational modifications and protein complexes and networks. Nowadays, the focus of quantitative proteomics shifted from the relative determination of proteins (ie, differential expression between two or more cellular states) to absolute quantity determination, required for a more-thorough characterization of biological models and comprehension of the proteome dynamism, as well as for the search and validation of novel protein biomarkers. However, the physico-chemical environment of the analyte species affects strongly the ionization efficiency in most mass spectrometry (MS) types, which thereby require the use of specially designed standardization approaches to provide absolute quantifications. Most common of such approaches nowadays include (i) the use of stable isotope-labeled peptide standards, isotopologues to the target proteotypic peptides expected after tryptic digestion of the target protein; (ii) use of stable isotope-labeled protein standards to compensate for sample preparation, sample loss, and proteolysis steps; (iii) isobaric reagents, which after fragmentation in the MS/MS analysis provide a final detectable mass shift, can be used to tag both analyte and standard samples; (iv) label-free approaches in which the absolute quantitative data are not obtained through the use of any kind of labeling, but from computational normalization of the raw data and adequate standards; (v) elemental mass spectrometry-based workflows able to provide directly absolute quantification of peptides/proteins that contain an ICP-detectable element. A critical insight from the Analytical Chemistry perspective of the different standardization approaches and their combinations used so far for absolute quantitative MS-based (molecular and
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A Quantitative Proteomics Approach to Clinical Research with Non-Traditional Samples
Licier, Rígel; Miranda, Eric; Serrano, Horacio
2016-01-01
The proper handling of samples to be analyzed by mass spectrometry (MS) can guarantee excellent results and a greater depth of analysis when working in quantitative proteomics. This is critical when trying to assess non-traditional sources such as ear wax, saliva, vitreous humor, aqueous humor, tears, nipple aspirate fluid, breast milk/colostrum, cervical-vaginal fluid, nasal secretions, bronco-alveolar lavage fluid, and stools. We intend to provide the investigator with relevant aspects of quantitative proteomics and to recognize the most recent clinical research work conducted with atypical samples and analyzed by quantitative proteomics. Having as reference the most recent and different approaches used with non-traditional sources allows us to compare new strategies in the development of novel experimental models. On the other hand, these references help us to contribute significantly to the understanding of the proportions of proteins in different proteomes of clinical interest and may lead to potential advances in the emerging field of precision medicine. PMID:28248241
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A Quantitative Proteomics Approach to Clinical Research with Non-Traditional Samples.
Licier, Rígel; Miranda, Eric; Serrano, Horacio
2016-10-17
The proper handling of samples to be analyzed by mass spectrometry (MS) can guarantee excellent results and a greater depth of analysis when working in quantitative proteomics. This is critical when trying to assess non-traditional sources such as ear wax, saliva, vitreous humor, aqueous humor, tears, nipple aspirate fluid, breast milk/colostrum, cervical-vaginal fluid, nasal secretions, bronco-alveolar lavage fluid, and stools. We intend to provide the investigator with relevant aspects of quantitative proteomics and to recognize the most recent clinical research work conducted with atypical samples and analyzed by quantitative proteomics. Having as reference the most recent and different approaches used with non-traditional sources allows us to compare new strategies in the development of novel experimental models. On the other hand, these references help us to contribute significantly to the understanding of the proportions of proteins in different proteomes of clinical interest and may lead to potential advances in the emerging field of precision medicine.
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Comparative and Quantitative Global Proteomics Approaches: An Overview
Deracinois, Barbara; Flahaut, Christophe; Duban-Deweer, Sophie; Karamanos, Yannis
2013-01-01
Proteomics became a key tool for the study of biological systems. The comparison between two different physiological states allows unravelling the cellular and molecular mechanisms involved in a biological process. Proteomics can confirm the presence of proteins suggested by their mRNA content and provides a direct measure of the quantity present in a cell. Global and targeted proteomics strategies can be applied. Targeted proteomics strategies limit the number of features that will be monitored and then optimise the methods to obtain the highest sensitivity and throughput for a huge amount of samples. The advantage of global proteomics strategies is that no hypothesis is required, other than a measurable difference in one or more protein species between the samples. Global proteomics methods attempt to separate quantify and identify all the proteins from a given sample. This review highlights only the different techniques of separation and quantification of proteins and peptides, in view of a comparative and quantitative global proteomics analysis. The in-gel and off-gel quantification of proteins will be discussed as well as the corresponding mass spectrometry technology. The overview is focused on the widespread techniques while keeping in mind that each approach is modular and often recovers the other. PMID:28250403
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Quantitative proteomics in cardiovascular research: global and targeted strategies
Shen, Xiaomeng; Young, Rebeccah; Canty, John M.; Qu, Jun
2014-01-01
Extensive technical advances in the past decade have substantially expanded quantitative proteomics in cardiovascular research. This has great promise for elucidating the mechanisms of cardiovascular diseases (CVD) and the discovery of cardiac biomarkers used for diagnosis and treatment evaluation. Global and targeted proteomics are the two major avenues of quantitative proteomics. While global approaches enable unbiased discovery of altered proteins via relative quantification at the proteome level, targeted techniques provide higher sensitivity and accuracy, and are capable of multiplexed absolute quantification in numerous clinical/biological samples. While promising, technical challenges need to be overcome to enable full utilization of these techniques in cardiovascular medicine. Here we discuss recent advances in quantitative proteomics and summarize applications in cardiovascular research with an emphasis on biomarker discovery and elucidating molecular mechanisms of disease. We propose the integration of global and targeted strategies as a high-throughput pipeline for cardiovascular proteomics. Targeted approaches enable rapid, extensive validation of biomarker candidates discovered by global proteomics. These approaches provide a promising alternative to immunoassays and other low-throughput means currently used for limited validation. PMID:24920501
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Bilić, Petra; Guillemin, Nicolas; Kovačević, Alan; Beer Ljubić, Blanka; Jović, Ines; Galan, Asier; Eckersall, Peter David; Burchmore, Richard; Mrljak, Vladimir
2018-05-15
Idiopathic dilated cardiomyopathy (iDCM) is a primary myocardial disorder with an unknown aetiology, characterized by reduced contractility and ventricular dilation of the left or both ventricles. Naturally occurring canine iDCM was used herein to identify serum proteomic signature of the disease compared to the healthy state, providing an insight into underlying mechanisms and revealing proteins with biomarker potential. To achieve this, we used high-throughput label-based quantitative LC-MS/MS proteomics approach and bioinformatics analysis of the in silico inferred interactome protein network created from the initial list of differential proteins. To complement the proteomic analysis, serum biochemical parameters and levels of know biomarkers of cardiac function were measured. Several proteins with biomarker potential were identified, such as inter-alpha-trypsin inhibitor heavy chain H4, microfibril-associated glycoprotein 4 and apolipoprotein A-IV, which were validated using an independent method (Western blotting) and showed high specificity and sensitivity according to the receiver operating characteristic curve analysis. Bioinformatics analysis revealed involvement of different pathways in iDCM, such as complement cascade activation, lipoprotein particles dynamics, elastic fibre formation, GPCR signalling and respiratory electron transport chain. Idiopathic dilated cardiomyopathy is a severe primary myocardial disease of unknown cause, affecting both humans and dogs. This study is a contribution to the canine heart disease research by means of proteomic and bioinformatic state of the art analyses, following similar approach in human iDCM research. Importantly, we used serum as non-invasive and easily accessible biological source of information and contributed to the scarce data on biofluid proteome research on this topic. Bioinformatics analysis revealed biological pathways modulated in canine iDCM with potential of further targeted research. Also, several
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Design and analysis issues in quantitative proteomics studies.
Karp, Natasha A; Lilley, Kathryn S
2007-09-01
Quantitative proteomics is the comparison of distinct proteomes which enables the identification of protein species which exhibit changes in expression or post-translational state in response to a given stimulus. Many different quantitative techniques are being utilized and generate large datasets. Independent of the technique used, these large datasets need robust data analysis to ensure valid conclusions are drawn from such studies. Approaches to address the problems that arise with large datasets are discussed to give insight into the types of statistical analyses of data appropriate for the various experimental strategies that can be employed by quantitative proteomic studies. This review also highlights the importance of employing a robust experimental design and highlights various issues surrounding the design of experiments. The concepts and examples discussed within will show how robust design and analysis will lead to confident results that will ensure quantitative proteomics delivers.
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[Methods of quantitative proteomics].
Kopylov, A T; Zgoda, V G
2007-01-01
In modern science proteomic analysis is inseparable from other fields of systemic biology. Possessing huge resources quantitative proteomics operates colossal information on molecular mechanisms of life. Advances in proteomics help researchers to solve complex problems of cell signaling, posttranslational modification, structure and functional homology of proteins, molecular diagnostics etc. More than 40 various methods have been developed in proteomics for quantitative analysis of proteins. Although each method is unique and has certain advantages and disadvantages all these use various isotope labels (tags). In this review we will consider the most popular and effective methods employing both chemical modifications of proteins and also metabolic and enzymatic methods of isotope labeling.
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Quantitative proteomics in biological research.
Wilm, Matthias
2009-10-01
Proteomics has enabled the direct investigation of biological material, at first through the analysis of individual proteins, then of lysates from cell cultures, and finally of extracts from tissues and biopsies from entire organisms. Its latest manifestation - quantitative proteomics - allows deeper insight into biological systems. This article reviews the different methods used to extract quantitative information from mass spectra. It follows the technical developments aimed toward global proteomics, the attempt to characterize every expressed protein in a cell by at least one peptide. When applications of the technology are discussed, the focus is placed on yeast biology. In particular, differential quantitative proteomics, the comparison between an experiment and its control, is very discriminating for proteins involved in the process being studied. When trying to understand biological processes on a molecular level, differential quantitative proteomics tends to give a clearer picture than global transcription analyses. As a result, MS has become an even more indispensable tool for biochemically motivated biological research.
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[Progress in stable isotope labeled quantitative proteomics methods].
Zhou, Yuan; Shan, Yichu; Zhang, Lihua; Zhang, Yukui
2013-06-01
Quantitative proteomics is an important research field in post-genomics era. There are two strategies for proteome quantification: label-free methods and stable isotope labeling methods which have become the most important strategy for quantitative proteomics at present. In the past few years, a number of quantitative methods have been developed, which support the fast development in biology research. In this work, we discuss the progress in the stable isotope labeling methods for quantitative proteomics including relative and absolute quantitative proteomics, and then give our opinions on the outlook of proteome quantification methods.
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Guidelines for reporting quantitative mass spectrometry based experiments in proteomics.
Martínez-Bartolomé, Salvador; Deutsch, Eric W; Binz, Pierre-Alain; Jones, Andrew R; Eisenacher, Martin; Mayer, Gerhard; Campos, Alex; Canals, Francesc; Bech-Serra, Joan-Josep; Carrascal, Montserrat; Gay, Marina; Paradela, Alberto; Navajas, Rosana; Marcilla, Miguel; Hernáez, María Luisa; Gutiérrez-Blázquez, María Dolores; Velarde, Luis Felipe Clemente; Aloria, Kerman; Beaskoetxea, Jabier; Medina-Aunon, J Alberto; Albar, Juan P
2013-12-16
Mass spectrometry is already a well-established protein identification tool and recent methodological and technological developments have also made possible the extraction of quantitative data of protein abundance in large-scale studies. Several strategies for absolute and relative quantitative proteomics and the statistical assessment of quantifications are possible, each having specific measurements and therefore, different data analysis workflows. The guidelines for Mass Spectrometry Quantification allow the description of a wide range of quantitative approaches, including labeled and label-free techniques and also targeted approaches such as Selected Reaction Monitoring (SRM). The HUPO Proteomics Standards Initiative (HUPO-PSI) has invested considerable efforts to improve the standardization of proteomics data handling, representation and sharing through the development of data standards, reporting guidelines, controlled vocabularies and tooling. In this manuscript, we describe a key output from the HUPO-PSI-namely the MIAPE Quant guidelines, which have developed in parallel with the corresponding data exchange format mzQuantML [1]. The MIAPE Quant guidelines describe the HUPO-PSI proposal concerning the minimum information to be reported when a quantitative data set, derived from mass spectrometry (MS), is submitted to a database or as supplementary information to a journal. The guidelines have been developed with input from a broad spectrum of stakeholders in the proteomics field to represent a true consensus view of the most important data types and metadata, required for a quantitative experiment to be analyzed critically or a data analysis pipeline to be reproduced. It is anticipated that they will influence or be directly adopted as part of journal guidelines for publication and by public proteomics databases and thus may have an impact on proteomics laboratories across the world. This article is part of a Special Issue entitled: Standardization and
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Quantitative proteomics in Giardia duodenalis-Achievements and challenges.
Emery, Samantha J; Lacey, Ernest; Haynes, Paul A
2016-08-01
Giardia duodenalis (syn. G. lamblia and G. intestinalis) is a protozoan parasite of vertebrates and a major contributor to the global burden of diarrheal diseases and gastroenteritis. The publication of multiple genome sequences in the G. duodenalis species complex has provided important insights into parasite biology, and made post-genomic technologies, including proteomics, significantly more accessible. The aims of proteomics are to identify and quantify proteins present in a cell, and assign functions to them within the context of dynamic biological systems. In Giardia, proteomics in the post-genomic era has transitioned from reliance on gel-based systems to utilisation of a diverse array of techniques based on bottom-up LC-MS/MS technologies. Together, these have generated crucial foundations for subcellular proteomes, elucidated intra- and inter-assemblage isolate variation, and identified pathways and markers in differentiation, host-parasite interactions and drug resistance. However, in Giardia, proteomics remains an emerging field, with considerable shortcomings evident from the published research. These include a bias towards assemblage A, a lack of emphasis on quantitative analytical techniques, and limited information on post-translational protein modifications. Additionally, there are multiple areas of research for which proteomic data is not available to add value to published transcriptomic data. The challenge of amalgamating data in the systems biology paradigm necessitates the further generation of large, high-quality quantitative datasets to accurately model parasite biology. This review surveys the current proteomic research available for Giardia and evaluates their technical and quantitative approaches, while contextualising their biological insights into parasite pathology, isolate variation and eukaryotic evolution. Finally, we propose areas of priority for the generation of future proteomic data to explore fundamental questions in Giardia
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Advances in Quantitative Proteomics of Microbes and Microbial Communities
NASA Astrophysics Data System (ADS)
Waldbauer, J.; Zhang, L.; Rizzo, A. I.
2015-12-01
Quantitative measurements of gene expression are key to developing a mechanistic, predictive understanding of how microbial metabolism drives many biogeochemical fluxes and responds to environmental change. High-throughput RNA-sequencing can afford a wealth of information about transcript-level expression patterns, but it is becoming clear that expression dynamics are often very different at the protein level where biochemistry actually occurs. These divergent dynamics between levels of biological organization necessitate quantitative proteomic measurements to address many biogeochemical questions. The protein-level expression changes that underlie shifts in the magnitude, or even the direction, of metabolic and biogeochemical fluxes can be quite subtle and test the limits of current quantitative proteomics techniques. Here we describe methodologies for high-precision, whole-proteome quantification that are applicable to both model organisms of biogeochemical interest that may not be genetically tractable, and to complex community samples from natural environments. Employing chemical derivatization of peptides with multiple isotopically-coded tags, this strategy is rapid and inexpensive, can be implemented on a wide range of mass spectrometric instrumentation, and is relatively insensitive to chromatographic variability. We demonstrate the utility of this quantitative proteomics approach in application to both isolates and natural communities of sulfur-metabolizing and photosynthetic microbes.
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Clinical veterinary proteomics: Techniques and approaches to decipher the animal plasma proteome.
Ghodasara, P; Sadowski, P; Satake, N; Kopp, S; Mills, P C
2017-12-01
Over the last two decades, technological advancements in the field of proteomics have advanced our understanding of the complex biological systems of living organisms. Techniques based on mass spectrometry (MS) have emerged as powerful tools to contextualise existing genomic information and to create quantitative protein profiles from plasma, tissues or cell lines of various species. Proteomic approaches have been used increasingly in veterinary science to investigate biological processes responsible for growth, reproduction and pathological events. However, the adoption of proteomic approaches by veterinary investigators lags behind that of researchers in the human medical field. Furthermore, in contrast to human proteomics studies, interpretation of veterinary proteomic data is difficult due to the limited protein databases available for many animal species. This review article examines the current use of advanced proteomics techniques for evaluation of animal health and welfare and covers the current status of clinical veterinary proteomics research, including successful protein identification and data interpretation studies. It includes a description of an emerging tool, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS), available on selected mass spectrometry instruments. This newly developed data acquisition technique combines advantages of discovery and targeted proteomics approaches, and thus has the potential to advance the veterinary proteomics field by enhancing identification and reproducibility of proteomics data. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Quantitative proteomics in the field of microbiology.
Otto, Andreas; Becher, Dörte; Schmidt, Frank
2014-03-01
Quantitative proteomics has become an indispensable analytical tool for microbial research. Modern microbial proteomics covers a wide range of topics in basic and applied research from in vitro characterization of single organisms to unravel the physiological implications of stress/starvation to description of the proteome content of a cell at a given time. With the techniques available, ranging from classical gel-based procedures to modern MS-based quantitative techniques, including metabolic and chemical labeling, as well as label-free techniques, quantitative proteomics is today highly successful in sophisticated settings of high complexity such as host-pathogen interactions, mixed microbial communities, and microbial metaproteomics. In this review, we will focus on the vast range of techniques practically applied in current research with an introduction of the workflows used for quantitative comparisons, a description of the advantages/disadvantages of the various methods, reference to hallmark publications and presentation of applications in current microbial research. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Ohtsuki, Sumio; Hirayama, Mio; Ito, Shingo; Uchida, Yasuo; Tachikawa, Masanori; Terasaki, Tetsuya
2014-06-01
The blood-brain barrier (BBB) is formed by brain capillary endothelial cells linked together via complex tight junctions, and serves to prevent entry of drugs into the brain. Multiple transporters are expressed at the BBB, where they control exchange of materials between the circulating blood and brain interstitial fluid, thereby supporting and protecting the CNS. An understanding of the BBB is necessary for efficient development of CNS-acting drugs and to identify potential drug targets for treatment of CNS diseases. Quantitative targeted proteomics can provide detailed information on protein expression levels at the BBB. The present review highlights the latest applications of quantitative targeted proteomics in BBB research, specifically to evaluate species and in vivo-in vitro differences, and to reconstruct in vivo transport activity. Such a BBB quantitative proteomics approach can be considered as pharmacoproteomics.
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Will Quantitative Proteomics Redefine Some of the Key Concepts in Skeletal Muscle Physiology?
Gizak, Agnieszka; Rakus, Dariusz
2016-01-11
Molecular and cellular biology methodology is traditionally based on the reasoning called "the mechanistic explanation". In practice, this means identifying and selecting correlations between biological processes which result from our manipulation of a biological system. In theory, a successful application of this approach requires precise knowledge about all parameters of a studied system. However, in practice, due to the systems' complexity, this requirement is rarely, if ever, accomplished. Typically, it is limited to a quantitative or semi-quantitative measurements of selected parameters (e.g., concentrations of some metabolites), and a qualitative or semi-quantitative description of expression/post-translational modifications changes within selected proteins. A quantitative proteomics approach gives a possibility of quantitative characterization of the entire proteome of a biological system, in the context of the titer of proteins as well as their post-translational modifications. This enables not only more accurate testing of novel hypotheses but also provides tools that can be used to verify some of the most fundamental dogmas of modern biology. In this short review, we discuss some of the consequences of using quantitative proteomics to verify several key concepts in skeletal muscle physiology.
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To label or not to label: applications of quantitative proteomics in neuroscience research.
Filiou, Michaela D; Martins-de-Souza, Daniel; Guest, Paul C; Bahn, Sabine; Turck, Christoph W
2012-02-01
Proteomics has provided researchers with a sophisticated toolbox of labeling-based and label-free quantitative methods. These are now being applied in neuroscience research where they have already contributed to the elucidation of fundamental mechanisms and the discovery of candidate biomarkers. In this review, we evaluate and compare labeling-based and label-free quantitative proteomic techniques for applications in neuroscience research. We discuss the considerations required for the analysis of brain and central nervous system specimens, the experimental design of quantitative proteomic workflows as well as the feasibility, advantages, and disadvantages of the available techniques for neuroscience-oriented questions. Furthermore, we assess the use of labeled standards as internal controls for comparative studies in humans and review applications of labeling-based and label-free mass spectrometry approaches in relevant model organisms and human subjects. Providing a comprehensive guide of feasible and meaningful quantitative proteomic methodologies for neuroscience research is crucial not only for overcoming current limitations but also for gaining useful insights into brain function and translating proteomics from bench to bedside. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Current trends in quantitative proteomics - an update.
Li, H; Han, J; Pan, J; Liu, T; Parker, C E; Borchers, C H
2017-05-01
Proteins can provide insights into biological processes at the functional level, so they are very promising biomarker candidates. The quantification of proteins in biological samples has been routinely used for the diagnosis of diseases and monitoring the treatment. Although large-scale protein quantification in complex samples is still a challenging task, a great amount of effort has been made to advance the technologies that enable quantitative proteomics. Seven years ago, in 2009, we wrote an article about the current trends in quantitative proteomics. In writing this current paper, we realized that, today, we have an even wider selection of potential tools for quantitative proteomics. These tools include new derivatization reagents, novel sampling formats, new types of analyzers and scanning techniques, and recently developed software to assist in assay development and data analysis. In this review article, we will discuss these innovative methods, and their current and potential applications in proteomics. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.
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Simulated linear test applied to quantitative proteomics.
Pham, T V; Jimenez, C R
2016-09-01
Omics studies aim to find significant changes due to biological or functional perturbation. However, gene and protein expression profiling experiments contain inherent technical variation. In discovery proteomics studies where the number of samples is typically small, technical variation plays an important role because it contributes considerably to the observed variation. Previous methods place both technical and biological variations in tightly integrated mathematical models that are difficult to adapt for different technological platforms. Our aim is to derive a statistical framework that allows the inclusion of a wide range of technical variability. We introduce a new method called the simulated linear test, or the s-test, that is easy to implement and easy to adapt for different models of technical variation. It generates virtual data points from the observed values according to a pre-defined technical distribution and subsequently employs linear modeling for significance analysis. We demonstrate the flexibility of the proposed approach by deriving a new significance test for quantitative discovery proteomics for which missing values have been a major issue for traditional methods such as the t-test. We evaluate the result on two label-free (phospho) proteomics datasets based on ion-intensity quantitation. Available at http://www.oncoproteomics.nl/software/stest.html : t.pham@vumc.nl. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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Rigbolt, Kristoffer T G; Vanselow, Jens T; Blagoev, Blagoy
2011-08-01
Recent technological advances have made it possible to identify and quantify thousands of proteins in a single proteomics experiment. As a result of these developments, the analysis of data has become the bottleneck of proteomics experiment. To provide the proteomics community with a user-friendly platform for comprehensive analysis, inspection and visualization of quantitative proteomics data we developed the Graphical Proteomics Data Explorer (GProX)(1). The program requires no special bioinformatics training, as all functions of GProX are accessible within its graphical user-friendly interface which will be intuitive to most users. Basic features facilitate the uncomplicated management and organization of large data sets and complex experimental setups as well as the inspection and graphical plotting of quantitative data. These are complemented by readily available high-level analysis options such as database querying, clustering based on abundance ratios, feature enrichment tests for e.g. GO terms and pathway analysis tools. A number of plotting options for visualization of quantitative proteomics data is available and most analysis functions in GProX create customizable high quality graphical displays in both vector and bitmap formats. The generic import requirements allow data originating from essentially all mass spectrometry platforms, quantitation strategies and software to be analyzed in the program. GProX represents a powerful approach to proteomics data analysis providing proteomics experimenters with a toolbox for bioinformatics analysis of quantitative proteomics data. The program is released as open-source and can be freely downloaded from the project webpage at http://gprox.sourceforge.net.
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Rigbolt, Kristoffer T. G.; Vanselow, Jens T.; Blagoev, Blagoy
2011-01-01
Recent technological advances have made it possible to identify and quantify thousands of proteins in a single proteomics experiment. As a result of these developments, the analysis of data has become the bottleneck of proteomics experiment. To provide the proteomics community with a user-friendly platform for comprehensive analysis, inspection and visualization of quantitative proteomics data we developed the Graphical Proteomics Data Explorer (GProX)1. The program requires no special bioinformatics training, as all functions of GProX are accessible within its graphical user-friendly interface which will be intuitive to most users. Basic features facilitate the uncomplicated management and organization of large data sets and complex experimental setups as well as the inspection and graphical plotting of quantitative data. These are complemented by readily available high-level analysis options such as database querying, clustering based on abundance ratios, feature enrichment tests for e.g. GO terms and pathway analysis tools. A number of plotting options for visualization of quantitative proteomics data is available and most analysis functions in GProX create customizable high quality graphical displays in both vector and bitmap formats. The generic import requirements allow data originating from essentially all mass spectrometry platforms, quantitation strategies and software to be analyzed in the program. GProX represents a powerful approach to proteomics data analysis providing proteomics experimenters with a toolbox for bioinformatics analysis of quantitative proteomics data. The program is released as open-source and can be freely downloaded from the project webpage at http://gprox.sourceforge.net. PMID:21602510
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Duan, Jicheng; Gaffrey, Matthew J.; Qian, Wei-Jun
Protein cysteine thiols play a crucial role in redox signaling, regulation of enzymatic activity and protein function, and maintaining redox homeostasis in living systems. The unique chemical reactivity of thiol groups makes cysteine susceptible to oxidative modifications by reactive oxygen and nitrogen species to form a broad array of reversible and irreversible protein post-translational modifications (PTMs). The reversible modifications in particular are one of the major components of redox signaling and are involved in regulation of various cellular processes under physiological and pathological conditions. The biological significance of these redox PTMs in health and diseases has been increasingly recognized. Herein,more » we review the recent advances of quantitative proteomic approaches for investigating redox PTMs in complex biological systems, including the general considerations of sample processing, various chemical or affinity enrichment strategies, and quantitative approaches. We also highlight a number of redox proteomic approaches that enable effective profiling of redox PTMs for addressing specific biological questions. Although some technological limitations remain, redox proteomics is paving the way towards a better understanding of redox signaling and regulation in human health and diseases.« less
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Woo, Jongmin; Han, Dohyun; Wang, Joseph Injae; Park, Joonho; Kim, Hyunsoo; Kim, Youngsoo
2017-09-01
The development of systematic proteomic quantification techniques in systems biology research has enabled one to perform an in-depth analysis of cellular systems. We have developed a systematic proteomic approach that encompasses the spectrum from global to targeted analysis on a single platform. We have applied this technique to an activated microglia cell system to examine changes in the intracellular and extracellular proteomes. Microglia become activated when their homeostatic microenvironment is disrupted. There are varying degrees of microglial activation, and we chose to focus on the proinflammatory reactive state that is induced by exposure to such stimuli as lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Using an improved shotgun proteomics approach, we identified 5497 proteins in the whole-cell proteome and 4938 proteins in the secretome that were associated with the activation of BV2 mouse microglia by LPS or IFN-γ. Of the differentially expressed proteins in stimulated microglia, we classified pathways that were related to immune-inflammatory responses and metabolism. Our label-free parallel reaction monitoring (PRM) approach made it possible to comprehensively measure the hyper-multiplex quantitative value of each protein by high-resolution mass spectrometry. Over 450 peptides that corresponded to pathway proteins and direct or indirect interactors via the STRING database were quantified by label-free PRM in a single run. Moreover, we performed a longitudinal quantification of secreted proteins during microglial activation, in which neurotoxic molecules that mediate neuronal cell loss in the brain are released. These data suggest that latent pathways that are associated with neurodegenerative diseases can be discovered by constructing and analyzing a pathway network model of proteins. Furthermore, this systematic quantification platform has tremendous potential for applications in large-scale targeted analyses. The proteomics data for
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Liquid Chromatography-Mass Spectrometry-based Quantitative Proteomics
DOE Office of Scientific and Technical Information (OSTI.GOV)
Xie, Fang; Liu, Tao; Qian, Weijun
2011-07-22
Liquid chromatography-mass spectrometry (LC-MS)-based quantitative proteomics has become increasingly applied for a broad range of biological applications due to growing capabilities for broad proteome coverage and good accuracy in quantification. Herein, we review the current LC-MS-based quantification methods with respect to their advantages and limitations, and highlight their potential applications.
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Proteomics approaches advance our understanding of plant self-incompatibility response.
Sankaranarayanan, Subramanian; Jamshed, Muhammad; Samuel, Marcus A
2013-11-01
Self-incompatibility (SI) in plants is a genetic mechanism that prevents self-fertilization and promotes out-crossing needed to maintain genetic diversity. SI has been classified into two broad categories: the gametophytic self-incompatibility (GSI) and the sporophytic self-incompatibility (SSI) based on the genetic mechanisms involved in 'self' pollen rejection. Recent proteomic approaches to identify potential candidates involved in SI have shed light onto a number of previously unidentified mechanisms required for SI response. SI proteome research has progressed from the use of isoelectric focusing in early days to the latest third-generation technique of comparative isobaric tag for relative and absolute quantitation (iTRAQ) used in recent times. We will focus on the proteome-based approaches used to study self-incompatibility (GSI and SSI), recent developments in the field of incompatibility research with emphasis on SSI and future prospects of using proteomic approaches to study self-incompatibility.
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Quantitative proteomics to study carbapenem resistance in Acinetobacter baumannii
Tiwari, Vishvanath; Tiwari, Monalisa
2014-01-01
Acinetobacter baumannii is an opportunistic pathogen causing pneumonia, respiratory infections and urinary tract infections. The prevalence of this lethal pathogen increases gradually in the clinical setup where it can grow on artificial surfaces, utilize ethanol as a carbon source. Moreover it resists desiccation. Carbapenems, a β-lactam, are the most commonly prescribed drugs against A. baumannii. Resistance against carbapenem has emerged in Acinetobacter baumannii which can create significant health problems and is responsible for high morbidity and mortality. With the development of quantitative proteomics, a considerable progress has been made in the study of carbapenem resistance of Acinetobacter baumannii. Recent updates showed that quantitative proteomics has now emerged as an important tool to understand the carbapenem resistance mechanism in Acinetobacter baumannii. Present review also highlights the complementary nature of different quantitative proteomic methods used to study carbapenem resistance and suggests to combine multiple proteomic methods for understanding the response to antibiotics by Acinetobacter baumannii. PMID:25309531
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Less label, more free: approaches in label-free quantitative mass spectrometry.
Neilson, Karlie A; Ali, Naveid A; Muralidharan, Sridevi; Mirzaei, Mehdi; Mariani, Michael; Assadourian, Gariné; Lee, Albert; van Sluyter, Steven C; Haynes, Paul A
2011-02-01
In this review we examine techniques, software, and statistical analyses used in label-free quantitative proteomics studies for area under the curve and spectral counting approaches. Recent advances in the field are discussed in an order that reflects a logical workflow design. Examples of studies that follow this design are presented to highlight the requirement for statistical assessment and further experiments to validate results from label-free quantitation. Limitations of label-free approaches are considered, label-free approaches are compared with labelling techniques, and forward-looking applications for label-free quantitative data are presented. We conclude that label-free quantitative proteomics is a reliable, versatile, and cost-effective alternative to labelled quantitation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Gunawardena, Harsha P.; Feltcher, Meghan E.; Wrobel, John A.; Gu, Sheng; Braunstein, Miriam; Chen, Xian
2015-01-01
The Mycobacterium tuberculosis (MTB) membrane is rich in antigens that are potential targets for diagnostics and the development of new vaccines. To better understand the mechanisms underlying MTB virulence and identify new targets for therapeutic intervention we investigated the differential composition of membrane proteomes between virulent M. tuberculosis H37Rv (MTB) and the Mycobacterium bovis BCG vaccine strain. To compare the membrane proteomes, we used LC-MS/MS analysis in combination with label-free quantitative (LFQ) proteomics, utilizing the area-under-curve (AUC) of the extracted ion chromatograms (XIC) of peptides obtained from m/z and retention time alignment of MS1 features. With this approach, we obtained relative abundance ratios for 2,203 identified membrane-associated proteins in high confidence. Of these proteins, 294 showed statistically significant differences of at least 2 fold, in relative abundance between MTB and BCG membrane fractions. Our comparative analysis detected several proteins associated with known genomic regions of difference between MTB and BCG as being absent, which validated the accuracy of our approach. In further support of our label-free quantitative data, we verified select protein differences by immunoblotting. To our knowledge we have generated the first comprehensive and high coverage profile of comparative membrane proteome changes between virulent MTB and its attenuated relative BCG, which helps elucidate the proteomic basis of the intrinsic virulence of the MTB pathogen. PMID:24093440
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Quantitative body fluid proteomics in medicine - A focus on minimal invasiveness.
Csősz, Éva; Kalló, Gergő; Márkus, Bernadett; Deák, Eszter; Csutak, Adrienne; Tőzsér, József
2017-02-05
Identification of new biomarkers specific for various pathological conditions is an important field in medical sciences. Body fluids have emerging potential in biomarker studies especially those which are continuously available and can be collected by non-invasive means. Changes in the protein composition of body fluids such as tears, saliva, sweat, etc. may provide information on both local and systemic conditions of medical relevance. In this review, our aim is to discuss the quantitative proteomics techniques used in biomarker studies, and to present advances in quantitative body fluid proteomics of non-invasively collectable body fluids with relevance to biomarker identification. The advantages and limitations of the widely used quantitative proteomics techniques are also presented. Based on the reviewed literature, we suggest an ideal pipeline for body fluid analyses aiming at biomarkers discoveries: starting from identification of biomarker candidates by shotgun quantitative proteomics or protein arrays, through verification of potential biomarkers by targeted mass spectrometry, to the antibody-based validation of biomarkers. The importance of body fluids as a rich source of biomarkers is discussed. Quantitative proteomics is a challenging part of proteomics applications. The body fluids collected by non-invasive means have high relevance in medicine; they are good sources for biomarkers used in establishing the diagnosis, follow up of disease progression and predicting high risk groups. The review presents the most widely used quantitative proteomics techniques in body fluid analysis and lists the potential biomarkers identified in tears, saliva, sweat, nasal mucus and urine for local and systemic diseases. Copyright © 2016 Elsevier B.V. All rights reserved.
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Method and platform standardization in MRM-based quantitative plasma proteomics.
Percy, Andrew J; Chambers, Andrew G; Yang, Juncong; Jackson, Angela M; Domanski, Dominik; Burkhart, Julia; Sickmann, Albert; Borchers, Christoph H
2013-12-16
There exists a growing demand in the proteomics community to standardize experimental methods and liquid chromatography-mass spectrometry (LC/MS) platforms in order to enable the acquisition of more precise and accurate quantitative data. This necessity is heightened by the evolving trend of verifying and validating candidate disease biomarkers in complex biofluids, such as blood plasma, through targeted multiple reaction monitoring (MRM)-based approaches with stable isotope-labeled standards (SIS). Considering the lack of performance standards for quantitative plasma proteomics, we previously developed two reference kits to evaluate the MRM with SIS peptide approach using undepleted and non-enriched human plasma. The first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). Here, these kits have been refined for practical use and then evaluated through intra- and inter-laboratory testing on 6 common LC/MS platforms. For an identical panel of 22 plasma proteins, similar concentrations were determined, regardless of the kit, instrument platform, and laboratory of analysis. These results demonstrate the value of the kit and reinforce the utility of standardized methods and protocols. The proteomics community needs standardized experimental protocols and quality control methods in order to improve the reproducibility of MS-based quantitative data. This need is heightened by the evolving trend for MRM-based validation of proposed disease biomarkers in complex biofluids such as blood plasma. We have developed two kits to assist in the inter- and intra-laboratory quality control of MRM experiments: the first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). In this paper, we report the use of these kits in intra- and inter-laboratory testing on 6 common LC/MS platforms. This
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Van Oudenhove, Laurence; Devreese, Bart
2013-06-01
Proteomics has evolved substantially since its early days, some 20 years ago. In this mini-review, we aim to provide an overview of general methodologies and more recent developments in mass spectrometric approaches used for relative and absolute quantitation of proteins. Enhancement of sensitivity of the mass spectrometers as well as improved sample preparation and protein fractionation methods are resulting in a more comprehensive analysis of proteomes. We also document some upcoming trends for quantitative proteomics such as the use of label-free quantification methods. Hopefully, microbiologists will continue to explore proteomics as a tool in their research to understand the adaptation of microorganisms to their ever changing environment. We encourage them to incorporate some of the described new developments in mass spectrometry to facilitate their analyses and improve the general knowledge of the fascinating world of microorganisms.
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Lavallée-Adam, Mathieu
2017-01-01
PSEA-Quant analyzes quantitative mass spectrometry-based proteomics datasets to identify enrichments of annotations contained in repositories such as the Gene Ontology and Molecular Signature databases. It allows users to identify the annotations that are significantly enriched for reproducibly quantified high abundance proteins. PSEA-Quant is available on the web and as a command-line tool. It is compatible with all label-free and isotopic labeling-based quantitative proteomics methods. This protocol describes how to use PSEA-Quant and interpret its output. The importance of each parameter as well as troubleshooting approaches are also discussed. PMID:27010334
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Clair, Geremy; Piehowski, Paul D.; Nicola, Teodora
Global proteomics approaches allow characterization of whole tissue lysates to an impressive depth. However, it is now increasingly recognized that to better understand the complexity of multicellular organisms, global protein profiling of specific spatially defined regions/substructures of tissues (i.e. spatially-resolved proteomics) is essential. Laser capture microdissection (LCM) enables microscopic isolation of defined regions of tissues preserving crucial spatial information. However, current proteomics workflows entail several manual sample preparation steps and are challenged by the microscopic mass-limited samples generated by LCM, and that impact measurement robustness, quantification, and throughput. Here, we coupled LCM with a fully automated sample preparation workflow thatmore » with a single manual step allows: protein extraction, tryptic digestion, peptide cleanup and LC-MS/MS analysis of proteomes from microdissected tissues. Benchmarking against the current state of the art in ultrasensitive global proteomic analysis, our approach demonstrated significant improvements in quantification and throughput. Using our LCM-SNaPP proteomics approach, we characterized to a depth of more than 3,400 proteins, the ontogeny of protein changes during normal lung development in laser capture microdissected alveolar tissue containing ~4,000 cells per sample. Importantly, the data revealed quantitative changes for 350 low abundance transcription factors and signaling molecules, confirming earlier transcript-level observations and defining seven modules of coordinated transcription factor/signaling molecule expression patterns, suggesting that a complex network of temporal regulatory control directs normal lung development with epigenetic regulation fine-tuning pre-natal developmental processes. Our LCM-proteomics approach facilitates efficient, spatially-resolved, ultrasensitive global proteomics analyses in high-throughput that will be enabling for several
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Lavallée-Adam, Mathieu; Yates, John R
2016-03-24
PSEA-Quant analyzes quantitative mass spectrometry-based proteomics datasets to identify enrichments of annotations contained in repositories such as the Gene Ontology and Molecular Signature databases. It allows users to identify the annotations that are significantly enriched for reproducibly quantified high abundance proteins. PSEA-Quant is available on the Web and as a command-line tool. It is compatible with all label-free and isotopic labeling-based quantitative proteomics methods. This protocol describes how to use PSEA-Quant and interpret its output. The importance of each parameter as well as troubleshooting approaches are also discussed. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.
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Clark, David J; Fondrie, William E; Liao, Zhongping; Hanson, Phyllis I; Fulton, Amy; Mao, Li; Yang, Austin J
2015-10-20
Exosomes are microvesicles of endocytic origin constitutively released by multiple cell types into the extracellular environment. With evidence that exosomes can be detected in the blood of patients with various malignancies, the development of a platform that uses exosomes as a diagnostic tool has been proposed. However, it has been difficult to truly define the exosome proteome due to the challenge of discerning contaminant proteins that may be identified via mass spectrometry using various exosome enrichment strategies. To better define the exosome proteome in breast cancer, we incorporated a combination of Tandem-Mass-Tag (TMT) quantitative proteomics approach and Support Vector Machine (SVM) cluster analysis of three conditioned media derived fractions corresponding to a 10 000g cellular debris pellet, a 100 000g crude exosome pellet, and an Optiprep enriched exosome pellet. The quantitative analysis identified 2 179 proteins in all three fractions, with known exosomal cargo proteins displaying at least a 2-fold enrichment in the exosome fraction based on the TMT protein ratios. Employing SVM cluster analysis allowed for the classification 251 proteins as "true" exosomal cargo proteins. This study provides a robust and vigorous framework for the future development of using exosomes as a potential multiprotein marker phenotyping tool that could be useful in breast cancer diagnosis and monitoring disease progression.
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Shen, Xiaomeng; Hu, Qiang; Li, Jun; Wang, Jianmin; Qu, Jun
2015-10-02
Comprehensive and accurate evaluation of data quality and false-positive biomarker discovery is critical to direct the method development/optimization for quantitative proteomics, which nonetheless remains challenging largely due to the high complexity and unique features of proteomic data. Here we describe an experimental null (EN) method to address this need. Because the method experimentally measures the null distribution (either technical or biological replicates) using the same proteomic samples, the same procedures and the same batch as the case-vs-contol experiment, it correctly reflects the collective effects of technical variability (e.g., variation/bias in sample preparation, LC-MS analysis, and data processing) and project-specific features (e.g., characteristics of the proteome and biological variation) on the performances of quantitative analysis. To show a proof of concept, we employed the EN method to assess the quantitative accuracy and precision and the ability to quantify subtle ratio changes between groups using different experimental and data-processing approaches and in various cellular and tissue proteomes. It was found that choices of quantitative features, sample size, experimental design, data-processing strategies, and quality of chromatographic separation can profoundly affect quantitative precision and accuracy of label-free quantification. The EN method was also demonstrated as a practical tool to determine the optimal experimental parameters and rational ratio cutoff for reliable protein quantification in specific proteomic experiments, for example, to identify the necessary number of technical/biological replicates per group that affords sufficient power for discovery. Furthermore, we assessed the ability of EN method to estimate levels of false-positives in the discovery of altered proteins, using two concocted sample sets mimicking proteomic profiling using technical and biological replicates, respectively, where the true
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Chen, Jin-Qiu; Wakefield, Lalage M; Goldstein, David J
2015-06-06
There is an emerging demand for the use of molecular profiling to facilitate biomarker identification and development, and to stratify patients for more efficient treatment decisions with reduced adverse effects. In the past decade, great strides have been made to advance genomic, transcriptomic and proteomic approaches to address these demands. While there has been much progress with these large scale approaches, profiling at the protein level still faces challenges due to limitations in clinical sample size, poor reproducibility, unreliable quantitation, and lack of assay robustness. A novel automated capillary nano-immunoassay (CNIA) technology has been developed. This technology offers precise and accurate measurement of proteins and their post-translational modifications using either charge-based or size-based separation formats. The system not only uses ultralow nanogram levels of protein but also allows multi-analyte analysis using a parallel single-analyte format for increased sensitivity and specificity. The high sensitivity and excellent reproducibility of this technology make it particularly powerful for analysis of clinical samples. Furthermore, the system can distinguish and detect specific protein post-translational modifications that conventional Western blot and other immunoassays cannot easily capture. This review will summarize and evaluate the latest progress to optimize the CNIA system for comprehensive, quantitative protein and signaling event characterization. It will also discuss how the technology has been successfully applied in both discovery research and clinical studies, for signaling pathway dissection, proteomic biomarker assessment, targeted treatment evaluation and quantitative proteomic analysis. Lastly, a comparison of this novel system with other conventional immuno-assay platforms is performed.
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El-Rami, Fadi; Nelson, Kristina; Xu, Ping
2017-01-01
Streptococcus sanguinis is a commensal and early colonizer of oral cavity as well as an opportunistic pathogen of infectious endocarditis. Extracting the soluble proteome of this bacterium provides deep insights about the physiological dynamic changes under different growth and stress conditions, thus defining “proteomic signatures” as targets for therapeutic intervention. In this protocol, we describe an experimentally verified approach to extract maximal cytoplasmic proteins from Streptococcus sanguinis SK36 strain. A combination of procedures was adopted that broke the thick cell wall barrier and minimized denaturation of the intracellular proteome, using optimized buffers and a sonication step. Extracted proteome was quantitated using Pierce BCA Protein Quantitation assay and protein bands were macroscopically assessed by Coomassie Blue staining. Finally, a high resolution detection of the extracted proteins was conducted through Synapt G2Si mass spectrometer, followed by label-free relative quantification via Progenesis QI. In conclusion, this pipeline for proteomic extraction and analysis of soluble proteins provides a fundamental tool in deciphering the biological complexity of Streptococcus sanguinis. PMID:29152022
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Alvarez, Sophie; Roy Choudhury, Swarup; Pandey, Sona
2014-03-07
Wheat is one of the most highly cultivated cereals in the world. Like other cultivated crops, wheat production is significantly affected by abiotic stresses such as drought. Multiple wheat varieties suitable for different geographical regions of the world have been developed that are adapted to different environmental conditions; however, the molecular basis of such adaptations remains unknown in most cases. We have compared the quantitative proteomics profile of the roots of two different wheat varieties, Nesser (drought-tolerant) and Opata (drought-sensitive), in the absence and presence of abscisic acid (ABA, as a proxy for drought). A labeling LC-based quantitative proteomics approach using iTRAQ was applied to elucidate the changes in protein abundance levels. Quantitative differences in protein levels were analyzed for the evaluation of inherent differences between the two varieties as well as the overall and variety-specific effect of ABA on the root proteome. This study reveals the most elaborate ABA-responsive root proteome identified to date in wheat. A large number of proteins exhibited inherently different expression levels between Nesser and Opata. Additionally, significantly higher numbers of proteins were ABA-responsive in Nesser roots compared with Opata roots. Furthermore, several proteins showed variety-specific regulation by ABA, suggesting their role in drought adaptation.
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Pfammatter, Sibylle; Bonneil, Eric; Thibault, Pierre
2016-12-02
Quantitative proteomics using isobaric reagent tandem mass tags (TMT) or isobaric tags for relative and absolute quantitation (iTRAQ) provides a convenient approach to compare changes in protein abundance across multiple samples. However, the analysis of complex protein digests by isobaric labeling can be undermined by the relative large proportion of co-selected peptide ions that lead to distorted reporter ion ratios and affect the accuracy and precision of quantitative measurements. Here, we investigated the use of high-field asymmetric waveform ion mobility spectrometry (FAIMS) in proteomic experiments to reduce sample complexity and improve protein quantification using TMT isobaric labeling. LC-FAIMS-MS/MS analyses of human and yeast protein digests led to significant reductions in interfering ions, which increased the number of quantifiable peptides by up to 68% while significantly improving the accuracy of abundance measurements compared to that with conventional LC-MS/MS. The improvement in quantitative measurements using FAIMS is further demonstrated for the temporal profiling of protein abundance of HEK293 cells following heat shock treatment.
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Li, Li; Luo, Zisheng; Huang, Xinhong; Zhang, Lu; Zhao, Pengyu; Ma, Hongyuan; Li, Xihong; Ban, Zhaojun; Liu, Xia
2015-04-29
To elucidate the mechanisms contributing to fruit responses to senescence and stressful environmental stimuli under low temperature (LT) and controlled atmosphere (CA) storage, a label-free quantitative proteomic investigation was conducted in strawberry (Fragaria ananassa, Duch. cv. 'Akihime'). Postharvest physiological quality traits including firmness, total soluble solids, total acidity, ascorbic acid and volatile production were characterized following storage under different conditions. The observed post-storage protein expression profiles may be associated with delayed senescence features in strawberry. A total of 454 proteins were identified in differentially treated strawberry fruits. Quantitative analysis, using normalized spectral counts, revealed 73 proteins common to all treatments, which formed three clusters in a hierarchical clustering analysis. The proteins spanned a range of functions in various metabolic pathways and networks involved in carbohydrate and energy metabolism, volatile biosynthesis, phenylpropanoid activity, stress response and protein synthesis, degradation and folding. After CA and LT storage, 16 (13) and 11 (17) proteins, respectively, were significantly increased (decreased) in abundance, while expression profile of 12 proteins was significantly changed by both CA and LT. To summarize, the differential variability of abundance in strawberry proteome, working in a cooperative manner, provided an overview of the biological processes that occurred during CA and LT storage. Controlled atmosphere storage at an optimal temperature is regarded to be an effective postharvest technology to delay fruit senescence and maintain fruit quality during shelf life. Nonetheless, little information on fruit proteomic changes under controlled atmosphere and/or low temperature storage is available. The significance of this paper is that it is the first study employing a label-free approach in the investigation of strawberry fruit response to
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Strigolactone-regulated proteins revealed by iTRAQ-based quantitative proteomics in Arabidopsis.
Li, Zhou; Czarnecki, Olaf; Chourey, Karuna; Yang, Jun; Tuskan, Gerald A; Hurst, Gregory B; Pan, Chongle; Chen, Jin-Gui
2014-03-07
Strigolactones (SLs) are a new class of plant hormones. In addition to acting as a key inhibitor of shoot branching, SLs stimulate seed germination of root parasitic plants and promote hyphal branching and root colonization of symbiotic arbuscular mycorrhizal fungi. They also regulate many other aspects of plant growth and development. At the transcription level, SL-regulated genes have been reported. However, nothing is known about the proteome regulated by this new class of plant hormones. A quantitative proteomics approach using an isobaric chemical labeling reagent, iTRAQ, to identify the proteome regulated by SLs in Arabidopsis seedlings is presented. It was found that SLs regulate the expression of about three dozen proteins that have not been previously assigned to SL pathways. These findings provide a new tool to investigate the molecular mechanism of action of SLs.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Li, Zhou; Adams, Rachel M; Chourey, Karuna
2012-01-01
A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification. Isobaricmore » chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. Based on the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.« less
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Statistical design of quantitative mass spectrometry-based proteomic experiments.
Oberg, Ann L; Vitek, Olga
2009-05-01
We review the fundamental principles of statistical experimental design, and their application to quantitative mass spectrometry-based proteomics. We focus on class comparison using Analysis of Variance (ANOVA), and discuss how randomization, replication and blocking help avoid systematic biases due to the experimental procedure, and help optimize our ability to detect true quantitative changes between groups. We also discuss the issues of pooling multiple biological specimens for a single mass analysis, and calculation of the number of replicates in a future study. When applicable, we emphasize the parallels between designing quantitative proteomic experiments and experiments with gene expression microarrays, and give examples from that area of research. We illustrate the discussion using theoretical considerations, and using real-data examples of profiling of disease.
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Missing Value Monitoring Enhances the Robustness in Proteomics Quantitation.
Matafora, Vittoria; Corno, Andrea; Ciliberto, Andrea; Bachi, Angela
2017-04-07
In global proteomic analysis, it is estimated that proteins span from millions to less than 100 copies per cell. The challenge of protein quantitation by classic shotgun proteomic techniques relies on the presence of missing values in peptides belonging to low-abundance proteins that lowers intraruns reproducibility affecting postdata statistical analysis. Here, we present a new analytical workflow MvM (missing value monitoring) able to recover quantitation of missing values generated by shotgun analysis. In particular, we used confident data-dependent acquisition (DDA) quantitation only for proteins measured in all the runs, while we filled the missing values with data-independent acquisition analysis using the library previously generated in DDA. We analyzed cell cycle regulated proteins, as they are low abundance proteins with highly dynamic expression levels. Indeed, we found that cell cycle related proteins are the major components of the missing values-rich proteome. Using the MvM workflow, we doubled the number of robustly quantified cell cycle related proteins, and we reduced the number of missing values achieving robust quantitation for proteins over ∼50 molecules per cell. MvM allows lower quantification variance among replicates for low abundance proteins with respect to DDA analysis, which demonstrates the potential of this novel workflow to measure low abundance, dynamically regulated proteins.
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Zhang, Kai; Tang, Chaohua; Liang, Xiaowei; Zhao, Qingyu; Zhang, Junmin
2018-01-10
Salbutamol, a selective β 2 -agonist, endangers the safety of animal products as a result of illegal use in food animals. In this study, an iTRAQ-based untargeted quantitative proteomic approach was applied to screen potential protein biomarkers in plasma of cattle before and after treatment with salbutamol for 21 days. A total of 62 plasma proteins were significantly affected by salbutamol treatment, which can be used as potential biomarkers to screen for the illegal use of salbutamol in beef cattle. Enzyme-linked immunosorbent assay measurements of five selected proteins demonstrated the reliability of iTRAQ-based proteomics in screening of candidate biomarkers among the plasma proteins. The plasma samples collected before and after salbutamol treatment were well-separated by principal component analysis (PCA) using the differentially expressed proteins. These results suggested that an iTRAQ-based untargeted quantitative proteomic strategy combined with PCA pattern recognition methods can discriminate differences in plasma protein profiles collected before and after salbutamol treatment.
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Guo, Jia; Nguyen, Amelia Y.; Dai, Ziyu; Su, Dian; Gaffrey, Matthew J.; Moore, Ronald J.; Jacobs, Jon M.; Monroe, Matthew E.; Smith, Richard D.; Koppenaal, David W.; Pakrasi, Himadri B.; Qian, Wei-Jun
2014-01-01
Reversible protein thiol oxidation is an essential regulatory mechanism of photosynthesis, metabolism, and gene expression in photosynthetic organisms. Herein, we present proteome-wide quantitative and site-specific profiling of in vivo thiol oxidation modulated by light/dark in the cyanobacterium Synechocystis sp. PCC 6803, an oxygenic photosynthetic prokaryote, using a resin-assisted thiol enrichment approach. Our proteomic approach integrates resin-assisted enrichment with isobaric tandem mass tag labeling to enable site-specific and quantitative measurements of reversibly oxidized thiols. The redox dynamics of ∼2,100 Cys-sites from 1,060 proteins under light, dark, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (a photosystem II inhibitor) conditions were quantified. In addition to relative quantification, the stoichiometry or percentage of oxidation (reversibly oxidized/total thiols) for ∼1,350 Cys-sites was also quantified. The overall results revealed broad changes in thiol oxidation in many key biological processes, including photosynthetic electron transport, carbon fixation, and glycolysis. Moreover, the redox sensitivity along with the stoichiometric data enabled prediction of potential functional Cys-sites for proteins of interest. The functional significance of redox-sensitive Cys-sites in NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, peroxiredoxin (AhpC/TSA family protein Sll1621), and glucose 6-phosphate dehydrogenase was further confirmed with site-specific mutagenesis and biochemical studies. Together, our findings provide significant insights into the broad redox regulation of photosynthetic organisms. PMID:25118246
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Determination of burn patient outcome by large-scale quantitative discovery proteomics
Finnerty, Celeste C.; Jeschke, Marc G.; Qian, Wei-Jun; Kaushal, Amit; Xiao, Wenzhong; Liu, Tao; Gritsenko, Marina A.; Moore, Ronald J.; Camp, David G.; Moldawer, Lyle L.; Elson, Constance; Schoenfeld, David; Gamelli, Richard; Gibran, Nicole; Klein, Matthew; Arnoldo, Brett; Remick, Daniel; Smith, Richard D.; Davis, Ronald; Tompkins, Ronald G.; Herndon, David N.
2013-01-01
Objective Emerging proteomics techniques can be used to establish proteomic outcome signatures and to identify candidate biomarkers for survival following traumatic injury. We applied high-resolution liquid chromatography-mass spectrometry (LC-MS) and multiplex cytokine analysis to profile the plasma proteome of survivors and non-survivors of massive burn injury to determine the proteomic survival signature following a major burn injury. Design Proteomic discovery study. Setting Five burn hospitals across the U.S. Patients Thirty-two burn patients (16 non-survivors and 16 survivors), 19–89 years of age, were admitted within 96 h of injury to the participating hospitals with burns covering >20% of the total body surface area and required at least one surgical intervention. Interventions None. Measurements and Main Results We found differences in circulating levels of 43 proteins involved in the acute phase response, hepatic signaling, the complement cascade, inflammation, and insulin resistance. Thirty-two of the proteins identified were not previously known to play a role in the response to burn. IL-4, IL-8, GM-CSF, MCP-1, and β2-microglobulin correlated well with survival and may serve as clinical biomarkers. Conclusions These results demonstrate the utility of these techniques for establishing proteomic survival signatures and for use as a discovery tool to identify candidate biomarkers for survival. This is the first clinical application of a high-throughput, large-scale LC-MS-based quantitative plasma proteomic approach for biomarker discovery for the prediction of patient outcome following burn, trauma or critical illness. PMID:23507713
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Quantitative trait loci mapping of the mouse plasma proteome (pQTL).
Holdt, Lesca M; von Delft, Annette; Nicolaou, Alexandros; Baumann, Sven; Kostrzewa, Markus; Thiery, Joachim; Teupser, Daniel
2013-02-01
A current challenge in the era of genome-wide studies is to determine the responsible genes and mechanisms underlying newly identified loci. Screening of the plasma proteome by high-throughput mass spectrometry (MALDI-TOF MS) is considered a promising approach for identification of metabolic and disease processes. Therefore, plasma proteome screening might be particularly useful for identifying responsible genes when combined with analysis of variation in the genome. Here, we describe a proteomic quantitative trait locus (pQTL) study of plasma proteome screens in an F(2) intercross of 455 mice mapped with 177 genetic markers across the genome. A total of 69 of 176 peptides revealed significant LOD scores (≥5.35) demonstrating strong genetic regulation of distinct components of the plasma proteome. Analyses were confirmed by mechanistic studies and MALDI-TOF/TOF, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the two strongest pQTLs: A pQTL for mass-to-charge ratio (m/z) 3494 (LOD 24.9, D11Mit151) was identified as the N-terminal 35 amino acids of hemoglobin subunit A (Hba) and caused by genetic variation in Hba. Another pQTL for m/z 8713 (LOD 36.4; D1Mit111) was caused by variation in apolipoprotein A2 (Apoa2) and cosegregated with HDL cholesterol. Taken together, we show that genome-wide plasma proteome profiling in combination with genome-wide genetic screening aids in the identification of causal genetic variants affecting abundance of plasma proteins.
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Quantitative proteomic analysis of microdissected oral epithelium for cancer biomarker discovery.
Xiao, Hua; Langerman, Alexander; Zhang, Yan; Khalid, Omar; Hu, Shen; Cao, Cheng-Xi; Lingen, Mark W; Wong, David T W
2015-11-01
Specific biomarkers are urgently needed for the detection and progression of oral cancer. The objective of this study was to discover cancer biomarkers from oral epithelium through utilizing high throughput quantitative proteomics approaches. Morphologically malignant, epithelial dysplasia, and adjacent normal epithelial tissues were laser capture microdissected (LCM) from 19 patients and used for proteomics analysis. Total proteins from each group were extracted, digested and then labelled with corresponding isobaric tags for relative and absolute quantitation (iTRAQ). Labelled peptides from each sample were combined and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) for protein identification and quantification. In total, 500 proteins were identified and 425 of them were quantified. When compared with adjacent normal oral epithelium, 17 and 15 proteins were consistently up-regulated or down-regulated in malignant and epithelial dysplasia, respectively. Half of these candidate biomarkers were discovered for oral cancer for the first time. Cornulin was initially confirmed in tissue protein extracts and was further validated in tissue microarray. Its presence in the saliva of oral cancer patients was also explored. Myoglobin and S100A8 were pre-validated by tissue microarray. These data demonstrated that the proteomic biomarkers discovered through this strategy are potential targets for oral cancer detection and salivary diagnostics. Copyright © 2015 Elsevier Ltd. All rights reserved.
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IsobariQ: software for isobaric quantitative proteomics using IPTL, iTRAQ, and TMT.
Arntzen, Magnus Ø; Koehler, Christian J; Barsnes, Harald; Berven, Frode S; Treumann, Achim; Thiede, Bernd
2011-02-04
Isobaric peptide labeling plays an important role in relative quantitative comparisons of proteomes. Isobaric labeling techniques utilize MS/MS spectra for relative quantification, which can be either based on the relative intensities of reporter ions in the low mass region (iTRAQ and TMT) or on the relative intensities of quantification signatures throughout the spectrum due to isobaric peptide termini labeling (IPTL). Due to the increased quantitative information found in MS/MS fragment spectra generated by the recently developed IPTL approach, new software was required to extract the quantitative information. IsobariQ was specifically developed for this purpose; however, support for the reporter ion techniques iTRAQ and TMT is also included. In addition, to address recently emphasized issues about heterogeneity of variance in proteomics data sets, IsobariQ employs the statistical software package R and variance stabilizing normalization (VSN) algorithms available therein. Finally, the functionality of IsobariQ is validated with data sets of experiments using 6-plex TMT and IPTL. Notably, protein substrates resulting from cleavage by proteases can be identified as shown for caspase targets in apoptosis.
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Ma, Hongyan; Delafield, Daniel G; Wang, Zhe; You, Jianlan; Wu, Si
2017-04-01
The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion. Graphical Abstract ᅟ.
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NASA Astrophysics Data System (ADS)
Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si
2017-04-01
The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.
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Data from quantitative label free proteomics analysis of rat spleen.
Dudekula, Khadar; Le Bihan, Thierry
2016-09-01
The dataset presented in this work has been obtained using a label-free quantitative proteomic analysis of rat spleen. A robust method for extraction of proteins from rat spleen tissue and LC-MS-MS analysis was developed using a urea and SDS-based buffer. Different fractionation methods were compared. A total of 3484 different proteins were identified from the pool of all experiments run in this study (a total of 2460 proteins with at least two peptides). A total of 1822 proteins were identified from nine non-fractionated pulse gels, 2288 proteins and 2864 proteins were identified by SDS-PAGE fractionation into three and five fractions respectively. The proteomics data are deposited in ProteomeXchange Consortium via PRIDE PXD003520, Progenesis and Maxquant output are presented in the supported information. The generated list of proteins under different regimes of fractionation allow assessing the nature of the identified proteins; variability in the quantitative analysis associated with the different sampling strategy and allow defining a proper number of replicates for future quantitative analysis.
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Quantitative Trait Loci Mapping of the Mouse Plasma Proteome (pQTL)
Holdt, Lesca M.; von Delft, Annette; Nicolaou, Alexandros; Baumann, Sven; Kostrzewa, Markus; Thiery, Joachim; Teupser, Daniel
2013-01-01
A current challenge in the era of genome-wide studies is to determine the responsible genes and mechanisms underlying newly identified loci. Screening of the plasma proteome by high-throughput mass spectrometry (MALDI-TOF MS) is considered a promising approach for identification of metabolic and disease processes. Therefore, plasma proteome screening might be particularly useful for identifying responsible genes when combined with analysis of variation in the genome. Here, we describe a proteomic quantitative trait locus (pQTL) study of plasma proteome screens in an F2 intercross of 455 mice mapped with 177 genetic markers across the genome. A total of 69 of 176 peptides revealed significant LOD scores (≥5.35) demonstrating strong genetic regulation of distinct components of the plasma proteome. Analyses were confirmed by mechanistic studies and MALDI-TOF/TOF, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the two strongest pQTLs: A pQTL for mass-to-charge ratio (m/z) 3494 (LOD 24.9, D11Mit151) was identified as the N-terminal 35 amino acids of hemoglobin subunit A (Hba) and caused by genetic variation in Hba. Another pQTL for m/z 8713 (LOD 36.4; D1Mit111) was caused by variation in apolipoprotein A2 (Apoa2) and cosegregated with HDL cholesterol. Taken together, we show that genome-wide plasma proteome profiling in combination with genome-wide genetic screening aids in the identification of causal genetic variants affecting abundance of plasma proteins. PMID:23172855
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Integrating cell biology and proteomic approaches in plants.
Takáč, Tomáš; Šamajová, Olga; Šamaj, Jozef
2017-10-03
Significant improvements of protein extraction, separation, mass spectrometry and bioinformatics nurtured advancements of proteomics during the past years. The usefulness of proteomics in the investigation of biological problems can be enhanced by integration with other experimental methods from cell biology, genetics, biochemistry, pharmacology, molecular biology and other omics approaches including transcriptomics and metabolomics. This review aims to summarize current trends integrating cell biology and proteomics in plant science. Cell biology approaches are most frequently used in proteomic studies investigating subcellular and developmental proteomes, however, they were also employed in proteomic studies exploring abiotic and biotic stress responses, vesicular transport, cytoskeleton and protein posttranslational modifications. They are used either for detailed cellular or ultrastructural characterization of the object subjected to proteomic study, validation of proteomic results or to expand proteomic data. In this respect, a broad spectrum of methods is employed to support proteomic studies including ultrastructural electron microscopy studies, histochemical staining, immunochemical localization, in vivo imaging of fluorescently tagged proteins and visualization of protein-protein interactions. Thus, cell biological observations on fixed or living cell compartments, cells, tissues and organs are feasible, and in some cases fundamental for the validation and complementation of proteomic data. Validation of proteomic data by independent experimental methods requires development of new complementary approaches. Benefits of cell biology methods and techniques are not sufficiently highlighted in current proteomic studies. This encouraged us to review most popular cell biology methods used in proteomic studies and to evaluate their relevance and potential for proteomic data validation and enrichment of purely proteomic analyses. We also provide examples of
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UNiquant, a program for quantitative proteomics analysis using stable isotope labeling.
Huang, Xin; Tolmachev, Aleksey V; Shen, Yulei; Liu, Miao; Huang, Lin; Zhang, Zhixin; Anderson, Gordon A; Smith, Richard D; Chan, Wing C; Hinrichs, Steven H; Fu, Kai; Ding, Shi-Jian
2011-03-04
Stable isotope labeling (SIL) methods coupled with nanoscale liquid chromatography and high resolution tandem mass spectrometry are increasingly useful for elucidation of the proteome-wide differences between multiple biological samples. Development of more effective programs for the sensitive identification of peptide pairs and accurate measurement of the relative peptide/protein abundance are essential for quantitative proteomic analysis. We developed and evaluated the performance of a new program, termed UNiquant, for analyzing quantitative proteomics data using stable isotope labeling. UNiquant was compared with two other programs, MaxQuant and Mascot Distiller, using SILAC-labeled complex proteome mixtures having either known or unknown heavy/light ratios. For the SILAC-labeled Jeko-1 cell proteome digests with known heavy/light ratios (H/L = 1:1, 1:5, and 1:10), UNiquant quantified a similar number of peptide pairs as MaxQuant for the H/L = 1:1 and 1:5 mixtures. In addition, UNiquant quantified significantly more peptides than MaxQuant and Mascot Distiller in the H/L = 1:10 mixtures. UNiquant accurately measured relative peptide/protein abundance without the need for postmeasurement normalization of peptide ratios, which is required by the other programs.
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UNiquant, a Program for Quantitative Proteomics Analysis Using Stable Isotope Labeling
Huang, Xin; Tolmachev, Aleksey V.; Shen, Yulei; Liu, Miao; Huang, Lin; Zhang, Zhixin; Anderson, Gordon A.; Smith, Richard D.; Chan, Wing C.; Hinrichs, Steven H.; Fu, Kai; Ding, Shi-Jian
2011-01-01
Stable isotope labeling (SIL) methods coupled with nanoscale liquid chromatography and high resolution tandem mass spectrometry are increasingly useful for elucidation of the proteome-wide differences between multiple biological samples. Development of more effective programs for the sensitive identification of peptide pairs and accurate measurement of the relative peptide/protein abundance are essential for quantitative proteomic analysis. We developed and evaluated the performance of a new program, termed UNiquant, for analyzing quantitative proteomics data using stable isotope labeling. UNiquant was compared with two other programs, MaxQuant and Mascot Distiller, using SILAC-labeled complex proteome mixtures having either known or unknown heavy/light ratios. For the SILAC-labeled Jeko-1 cell proteome digests with known heavy/light ratios (H/L = 1:1, 1:5, and 1:10), UNiquant quantified a similar number of peptide pairs as MaxQuant for the H/L = 1:1 and 1:5 mixtures. In addition, UNiquant quantified significantly more peptides than MaxQuant and Mascot Distiller in the H/L = 1:10 mixtures. UNiquant accurately measured relative peptide/protein abundance without the need for post-measurement normalization of peptide ratios, which is required by the other programs. PMID:21158445
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Liu, Kehui; Zhang, Jiyang; Fu, Bin; Xie, Hongwei; Wang, Yingchun; Qian, Xiaohong
2014-07-01
Precise protein quantification is essential in comparative proteomics. Currently, quantification bias is inevitable when using proteotypic peptide-based quantitative proteomics strategy for the differences in peptides measurability. To improve quantification accuracy, we proposed an "empirical rule for linearly correlated peptide selection (ERLPS)" in quantitative proteomics in our previous work. However, a systematic evaluation on general application of ERLPS in quantitative proteomics under diverse experimental conditions needs to be conducted. In this study, the practice workflow of ERLPS was explicitly illustrated; different experimental variables, such as, different MS systems, sample complexities, sample preparations, elution gradients, matrix effects, loading amounts, and other factors were comprehensively investigated to evaluate the applicability, reproducibility, and transferability of ERPLS. The results demonstrated that ERLPS was highly reproducible and transferable within appropriate loading amounts and linearly correlated response peptides should be selected for each specific experiment. ERLPS was used to proteome samples from yeast to mouse and human, and in quantitative methods from label-free to O18/O16-labeled and SILAC analysis, and enabled accurate measurements for all proteotypic peptide-based quantitative proteomics over a large dynamic range. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Gritsenko, Marina A.; Xu, Zhe; Liu, Tao
Comprehensive, quantitative information on abundances of proteins and their post-translational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labelling techniques combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification andmore » quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples, and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.« less
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Gritsenko, Marina A; Xu, Zhe; Liu, Tao; Smith, Richard D
2016-01-01
Comprehensive, quantitative information on abundances of proteins and their posttranslational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labeling techniques combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification and quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.
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A Routine 'Top-Down' Approach to Analysis of the Human Serum Proteome.
D'Silva, Arlene M; Hyett, Jon A; Coorssen, Jens R
2017-06-06
Serum provides a rich source of potential biomarker proteoforms. One of the major obstacles in analysing serum proteomes is detecting lower abundance proteins owing to the presence of hyper-abundant species (e.g., serum albumin and immunoglobulins). Although depletion methods have been used to address this, these can lead to the concomitant removal of non-targeted protein species, and thus raise issues of specificity, reproducibility, and the capacity for meaningful quantitative analyses. Altering the native stoichiometry of the proteome components may thus yield a more complex series of issues than dealing directly with the inherent complexity of the sample. Hence, here we targeted method refinements so as to ensure optimum resolution of serum proteomes via a top down two-dimensional gel electrophoresis (2DE) approach that enables the routine assessment of proteoforms and is fully compatible with subsequent mass spectrometric analyses. Testing included various fractionation and non-fractionation approaches. The data show that resolving 500 µg protein on 17 cm 3-10 non-linear immobilised pH gradient strips in the first dimension followed by second dimension resolution on 7-20% gradient gels with a combination of lithium dodecyl sulfate (LDS) and sodium dodecyl sulfate (SDS) detergents markedly improves the resolution and detection of proteoforms in serum. In addition, well established third dimension electrophoretic separations in combination with deep imaging further contributed to the best available resolution, detection, and thus quantitative top-down analysis of serum proteomes.
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Analysis of high accuracy, quantitative proteomics data in the MaxQB database.
Schaab, Christoph; Geiger, Tamar; Stoehr, Gabriele; Cox, Juergen; Mann, Matthias
2012-03-01
MS-based proteomics generates rapidly increasing amounts of precise and quantitative information. Analysis of individual proteomic experiments has made great strides, but the crucial ability to compare and store information across different proteome measurements still presents many challenges. For example, it has been difficult to avoid contamination of databases with low quality peptide identifications, to control for the inflation in false positive identifications when combining data sets, and to integrate quantitative data. Although, for example, the contamination with low quality identifications has been addressed by joint analysis of deposited raw data in some public repositories, we reasoned that there should be a role for a database specifically designed for high resolution and quantitative data. Here we describe a novel database termed MaxQB that stores and displays collections of large proteomics projects and allows joint analysis and comparison. We demonstrate the analysis tools of MaxQB using proteome data of 11 different human cell lines and 28 mouse tissues. The database-wide false discovery rate is controlled by adjusting the project specific cutoff scores for the combined data sets. The 11 cell line proteomes together identify proteins expressed from more than half of all human genes. For each protein of interest, expression levels estimated by label-free quantification can be visualized across the cell lines. Similarly, the expression rank order and estimated amount of each protein within each proteome are plotted. We used MaxQB to calculate the signal reproducibility of the detected peptides for the same proteins across different proteomes. Spearman rank correlation between peptide intensity and detection probability of identified proteins was greater than 0.8 for 64% of the proteome, whereas a minority of proteins have negative correlation. This information can be used to pinpoint false protein identifications, independently of peptide database
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Lebesgue, Nicolas; da Costa, Gonçalo; Ribeiro, Raquel Mesquita; Ribeiro-Silva, Cristina; Martins, Gabriel G; Matranga, Valeria; Scholten, Arjen; Cordeiro, Carlos; Heck, Albert J R; Santos, Romana
2016-04-14
Marine bioadhesives have unmatched performances in wet environments, being an inspiration for biomedical applications. In sea urchins specialized adhesive organs, tube feet, mediate reversible adhesion, being composed by a disc, producing adhesive and de-adhesive secretions, and a motile stem. After tube foot detachment, the secreted adhesive remains bound to the substratum as a footprint. Sea urchin adhesive is composed by proteins and sugars, but so far only one protein, Nectin, was shown to be over-expressed as a transcript in tube feet discs, suggesting its involvement in sea urchin adhesion. Here we use high-resolution quantitative mass-spectrometry to perform the first study combining the analysis of the differential proteome of an adhesive organ, with the proteome of its secreted adhesive. This strategy allowed us to identify 163 highly over-expressed disc proteins, specifically involved in sea urchin reversible adhesion; to find that 70% of the secreted adhesive components fall within five protein groups, involved in exocytosis and microbial protection; and to provide evidences that Nectin is not only highly expressed in tube feet discs but is an actual component of the adhesive. These results give an unprecedented insight into the molecular mechanisms underlying sea urchin adhesion, and opening new doors to develop wet-reliable, reversible, and ecological biomimetic adhesives. Sea urchins attach strongly but in a reversible manner to substratum, being a valuable source of inspiration for industrial and biomedical applications. Yet, the molecular mechanisms governing reversible adhesion are still poorly studied delaying the engineering of biomimetic adhesives. We used the latest mass spectrometry techniques to analyze the differential proteome of an adhesive organ and the proteome of its secreted adhesive, allowing us to uncover the key players in sea urchin reversible adhesion. We demonstrate, that Nectin, a protein previously pointed out as potentially
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Integrated Proteomic Approaches for Understanding Toxicity of Environmental Chemicals
To apply quantitative proteomic analysis to the evaluation of toxicity of environmental chemicals, we have developed an integrated proteomic technology platform. This platform has been applied to the analysis of the toxic effects and pathways of many important environmental chemi...
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A Targeted MRM Approach for Tempo-Spatial Proteomics Analyses.
Moradian, Annie; Porras-Yakushi, Tanya R; Sweredoski, Michael J; Hess, Sonja
2016-01-01
When deciding to perform a quantitative proteomics analysis, selectivity, sensitivity, and reproducibility are important criteria to consider. The use of multiple reaction monitoring (MRM) has emerged as a powerful proteomics technique in that regard since it avoids many of the problems typically observed in discovery-based analyses. A prerequisite for such a targeted approach is that the protein targets are known, either as a result of previous global proteomics experiments or because a specific hypothesis is to be tested. When guidelines that have been established in the pharmaceutical industry many decades ago are taken into account, setting up an MRM assay is relatively straightforward. Typically, proteotypic peptides with favorable mass spectrometric properties are synthesized with a heavy isotope for each protein that is to be monitored. Retention times and calibration curves are determined using triple-quadrupole mass spectrometers. The use of iRT peptide standards is both recommended and fully integrated into the bioinformatics pipeline. Digested biological samples are mixed with the heavy and iRT standards and quantified. Here we present a generic protocol for the development of an MRM assay.
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Martínez-Bussenius, Cristóbal; Navarro, Claudio A; Orellana, Luis; Paradela, Alberto; Jerez, Carlos A
2016-08-11
Acidithiobacillus ferrooxidans is used in industrial bioleaching of minerals to extract valuable metals. A. ferrooxidans strain ATCC 53993 is much more resistant to copper than other strains of this microorganism and it has been proposed that genes present in an exclusive genomic island (GI) of this strain would contribute to its extreme copper tolerance. ICPL (isotope-coded protein labeling) quantitative proteomics was used to study in detail the response of this bacterium to copper. A high overexpression of RND efflux systems and CusF copper chaperones, both present in the genome and the GI of strain ATCC 53993 was found. Also, changes in the levels of the respiratory system proteins such as AcoP and Rus copper binding proteins and several proteins with other predicted functions suggest that numerous metabolic changes are apparently involved in controlling the effects of the toxic metal on this acidophile. Using quantitative proteomics we overview the adaptation mechanisms that biomining acidophiles use to stand their harsh environment. The overexpression of several genes present in an exclusive genomic island strongly suggests the importance of the proteins coded in this DNA region in the high tolerance of A. ferrooxidans ATCC 53993 to metals. Copyright © 2016 Elsevier B.V. All rights reserved.
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Chemical proteomics approaches for identifying the cellular targets of natural products
Sieber, S. A.
2016-01-01
Covering: 2010 up to 2016 Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chemistry and biology today. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to understand protein function. In the context of natural products, chemical proteomics can be used to identify the protein binding partners or targets of small molecules in live cells. Here, we highlight recent examples of chemical probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chemical reactivity or via the introduction of photocrosslinkers), and can be applied “in situ” – in living systems rather than cell lysates. We also focus here on strategies that employ a click reaction, the copper-catalysed azide–alkyne cycloaddition reaction (CuAAC), to allow minimal functionalisation of natural product scaffolds with an alkyne or azide tag. We also discuss ‘competitive mode’ approaches that screen for natural products that compete with a well-characterised chemical probe for binding to a particular set of protein targets. Fuelled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quantitative proteomics to help define the true interacting partners of probes, and we highlight the opportunities this rapidly evolving technology provides in chemical proteomics. Finally, some of the limitations and challenges of chemical proteomics approaches are discussed. PMID:27098809
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Chemical proteomics approaches for identifying the cellular targets of natural products.
Wright, M H; Sieber, S A
2016-05-04
Covering: 2010 up to 2016Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chemistry and biology today. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to understand protein function. In the context of natural products, chemical proteomics can be used to identify the protein binding partners or targets of small molecules in live cells. Here, we highlight recent examples of chemical probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chemical reactivity or via the introduction of photocrosslinkers), and can be applied "in situ" - in living systems rather than cell lysates. We also focus here on strategies that employ a click reaction, the copper-catalysed azide-alkyne cycloaddition reaction (CuAAC), to allow minimal functionalisation of natural product scaffolds with an alkyne or azide tag. We also discuss 'competitive mode' approaches that screen for natural products that compete with a well-characterised chemical probe for binding to a particular set of protein targets. Fuelled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quantitative proteomics to help define the true interacting partners of probes, and we highlight the opportunities this rapidly evolving technology provides in chemical proteomics. Finally, some of the limitations and challenges of chemical proteomics approaches are discussed.
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Mahadevan, Chidambareswaren; Krishnan, Anu; Saraswathy, Gayathri G.; Surendran, Arun; Jaleel, Abdul; Sakuntala, Manjula
2016-01-01
Black pepper (Piper nigrum L.), a tropical spice crop of global acclaim, is susceptible to Phytophthora capsici, an oomycete pathogen which causes the highly destructive foot rot disease. A systematic understanding of this phytopathosystem has not been possible owing to lack of genome or proteome information. In this study, we explain an integrated transcriptome-assisted label-free quantitative proteomics pipeline to study the basal immune components of black pepper when challenged with P. capsici. We report a global identification of 532 novel leaf proteins from black pepper, of which 518 proteins were functionally annotated using BLAST2GO tool. A label-free quantitation of the protein datasets revealed 194 proteins common to diseased and control protein datasets of which 22 proteins showed significant up-regulation and 134 showed significant down-regulation. Ninety-three proteins were identified exclusively on P. capsici infected leaf tissues and 245 were expressed only in mock (control) infected samples. In-depth analysis of our data gives novel insights into the regulatory pathways of black pepper which are compromised during the infection. Differential down-regulation was observed in a number of critical pathways like carbon fixation in photosynthetic organism, cyano-amino acid metabolism, fructose, and mannose metabolism, glutathione metabolism, and phenylpropanoid biosynthesis. The proteomics results were validated with real-time qRT-PCR analysis. We were also able to identify the complete coding sequences for all the proteins of which few selected genes were cloned and sequence characterized for further confirmation. Our study is the first report of a quantitative proteomics dataset in black pepper which provides convincing evidence on the effectiveness of a transcriptome-based label-free proteomics approach for elucidating the host response to biotic stress in a non-model spice crop like P. nigrum, for which genome information is unavailable. Our dataset
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Mahadevan, Chidambareswaren; Krishnan, Anu; Saraswathy, Gayathri G; Surendran, Arun; Jaleel, Abdul; Sakuntala, Manjula
2016-01-01
Black pepper (Piper nigrum L.), a tropical spice crop of global acclaim, is susceptible to Phytophthora capsici, an oomycete pathogen which causes the highly destructive foot rot disease. A systematic understanding of this phytopathosystem has not been possible owing to lack of genome or proteome information. In this study, we explain an integrated transcriptome-assisted label-free quantitative proteomics pipeline to study the basal immune components of black pepper when challenged with P. capsici. We report a global identification of 532 novel leaf proteins from black pepper, of which 518 proteins were functionally annotated using BLAST2GO tool. A label-free quantitation of the protein datasets revealed 194 proteins common to diseased and control protein datasets of which 22 proteins showed significant up-regulation and 134 showed significant down-regulation. Ninety-three proteins were identified exclusively on P. capsici infected leaf tissues and 245 were expressed only in mock (control) infected samples. In-depth analysis of our data gives novel insights into the regulatory pathways of black pepper which are compromised during the infection. Differential down-regulation was observed in a number of critical pathways like carbon fixation in photosynthetic organism, cyano-amino acid metabolism, fructose, and mannose metabolism, glutathione metabolism, and phenylpropanoid biosynthesis. The proteomics results were validated with real-time qRT-PCR analysis. We were also able to identify the complete coding sequences for all the proteins of which few selected genes were cloned and sequence characterized for further confirmation. Our study is the first report of a quantitative proteomics dataset in black pepper which provides convincing evidence on the effectiveness of a transcriptome-based label-free proteomics approach for elucidating the host response to biotic stress in a non-model spice crop like P. nigrum, for which genome information is unavailable. Our dataset
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Lawless, Craig; Hubbard, Simon J.; Fan, Jun; Bessant, Conrad; Hermjakob, Henning; Jones, Andrew R.
2012-01-01
Abstract New methods for performing quantitative proteome analyses based on differential labeling protocols or label-free techniques are reported in the literature on an almost monthly basis. In parallel, a correspondingly vast number of software tools for the analysis of quantitative proteomics data has also been described in the literature and produced by private companies. In this article we focus on the review of some of the most popular techniques in the field and present a critical appraisal of several software packages available to process and analyze the data produced. We also describe the importance of community standards to support the wide range of software, which may assist researchers in the analysis of data using different platforms and protocols. It is intended that this review will serve bench scientists both as a useful reference and a guide to the selection and use of different pipelines to perform quantitative proteomics data analysis. We have produced a web-based tool (http://www.proteosuite.org/?q=other_resources) to help researchers find appropriate software for their local instrumentation, available file formats, and quantitative methodology. PMID:22804616
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Science, marketing and wishful thinking in quantitative proteomics.
Hackett, Murray
2008-11-01
In a recent editorial (J. Proteome Res. 2007, 6, 1633) and elsewhere questions have been raised regarding the lack of attention paid to good analytical practice with respect to the reporting of quantitative results in proteomics. Using those comments as a starting point, several issues are discussed that relate to the challenges involved in achieving adequate sampling with MS-based methods in order to generate valid data for large-scale studies. The discussion touches on the relationships that connect sampling depth and the power to detect protein abundance change, conflict of interest, and strategies to overcome bureaucratic obstacles that impede the use of peer-to-peer technologies for transfer and storage of large data files generated in such experiments.
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A Method for Label-Free, Differential Top-Down Proteomics.
Ntai, Ioanna; Toby, Timothy K; LeDuc, Richard D; Kelleher, Neil L
2016-01-01
Biomarker discovery in the translational research has heavily relied on labeled and label-free quantitative bottom-up proteomics. Here, we describe a new approach to biomarker studies that utilizes high-throughput top-down proteomics and is the first to offer whole protein characterization and relative quantitation within the same experiment. Using yeast as a model, we report procedures for a label-free approach to quantify the relative abundance of intact proteins ranging from 0 to 30 kDa in two different states. In this chapter, we describe the integrated methodology for the large-scale profiling and quantitation of the intact proteome by liquid chromatography-mass spectrometry (LC-MS) without the need for metabolic or chemical labeling. This recent advance for quantitative top-down proteomics is best implemented with a robust and highly controlled sample preparation workflow before data acquisition on a high-resolution mass spectrometer, and the application of a hierarchical linear statistical model to account for the multiple levels of variance contained in quantitative proteomic comparisons of samples for basic and clinical research.
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Scientific Approaches | Office of Cancer Clinical Proteomics Research
CPTAC employs two complementary scientific approaches, a "Targeting Genome to Proteome" (Targeting G2P) approach and a "Mapping Proteome to Genome" (Mapping P2G) approach, in order to address biological questions from data generated on a sample.
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Optimization of Statistical Methods Impact on Quantitative Proteomics Data.
Pursiheimo, Anna; Vehmas, Anni P; Afzal, Saira; Suomi, Tomi; Chand, Thaman; Strauss, Leena; Poutanen, Matti; Rokka, Anne; Corthals, Garry L; Elo, Laura L
2015-10-02
As tools for quantitative label-free mass spectrometry (MS) rapidly develop, a consensus about the best practices is not apparent. In the work described here we compared popular statistical methods for detecting differential protein expression from quantitative MS data using both controlled experiments with known quantitative differences for specific proteins used as standards as well as "real" experiments where differences in protein abundance are not known a priori. Our results suggest that data-driven reproducibility-optimization can consistently produce reliable differential expression rankings for label-free proteome tools and are straightforward in their application.
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Elamin, Ashraf; Titz, Bjoern; Dijon, Sophie; Merg, Celine; Geertz, Marcel; Schneider, Thomas; Martin, Florian; Schlage, Walter K; Frentzel, Stefan; Talamo, Fabio; Phillips, Blaine; Veljkovic, Emilija; Ivanov, Nikolai V; Vanscheeuwijck, Patrick; Peitsch, Manuel C; Hoeng, Julia
2016-08-11
Smoking is associated with several serious diseases, such as lung cancer and chronic obstructive pulmonary disease (COPD). Within our systems toxicology framework, we are assessing whether potential modified risk tobacco products (MRTP) can reduce smoking-related health risks compared to conventional cigarettes. In this article, we evaluated to what extent 2D-PAGE/MALDI MS/MS (2D-PAGE) can complement the iTRAQ LC-MS/MS results from a previously reported mouse inhalation study, in which we assessed a prototypic MRTP (pMRTP). Selected differentially expressed proteins identified by both LC-MS/MS and 2D-PAGE approaches were further verified using reverse-phase protein microarrays. LC-MS/MS captured the effects of cigarette smoke (CS) on the lung proteome more comprehensively than 2D-PAGE. However, an integrated analysis of both proteomics data sets showed that 2D-PAGE data complement the LC-MS/MS results by supporting the overall trend of lower effects of pMRTP aerosol than CS on the lung proteome. Biological effects of CS exposure supported by both methods included increases in immune-related, surfactant metabolism, proteasome, and actin cytoskeleton protein clusters. Overall, while 2D-PAGE has its value, especially as a complementary method for the analysis of effects on intact proteins, LC-MS/MS approaches will likely be the method of choice for proteome analysis in systems toxicology investigations. Quantitative proteomics is anticipated to play a growing role within systems toxicology assessment frameworks in the future. To further understand how different proteomics technologies can contribute to toxicity assessment, we conducted a quantitative proteomics analysis using 2D-PAGE and isobaric tag-based LC-MS/MS approaches and compared the results produced from the 2 approaches. Using a prototypic modified risk tobacco product (pMRTP) as our test item, we show compared with cigarette smoke, how 2D-PAGE results can complement and support LC-MS/MS data, demonstrating
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Orme, Rowan P; Gates, Monte A; Fricker-Gates, Rosemary A
2010-08-15
Cell transplantation using stem cell-derived neurons is commonly viewed as a candidate therapy for neurodegenerative diseases. However, methods for differentiating stem cells into homogenous populations of neurons suitable for transplant remain elusive. This suggests that there are as yet unknown signalling factors working in vivo to specify neuronal cell fate during development. These factors could be manipulated to better differentiate stem cells into neural populations useful for therapeutic transplantation. Here a quantitative proteomics approach is described for investigating cell signalling in the developing central nervous system (CNS), using the embryonic ventral mesencephalon as a model. Briefly, total protein was extracted from embryonic ventral midbrain tissue before, during and after the birth of dopaminergic neurons, and digested using trypsin. Two-dimensional liquid chromatography, coupled with tandem mass spectrometry, was then used to identify proteins from the tryptic peptides. Isobaric tagging for relative and absolute quantification (iTRAQ) reagents were used to label the tryptic peptides and facilitate relative quantitative analysis. The success of the experiment was confirmed by the identification of proteins known to be expressed in the developing ventral midbrain, as well as by Western blotting, and immunolabelling of embryonic tissue sections. This method of protein discovery improves upon previous attempts to identify novel signalling factors through microarray analysis. Importantly, the methods described here could be applied to virtually any aspect of development. (c) 2010 Elsevier B.V. All rights reserved.
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Quantitative and temporal proteome analysis of butyrate-treated colorectal cancer cells.
Tan, Hwee Tong; Tan, Sandra; Lin, Qingsong; Lim, Teck Kwang; Hew, Choy Leong; Chung, Maxey C M
2008-06-01
Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a short-chain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades. Previously we performed two-dimensional DIGE to identify differentially expressed proteins induced by 24-h butyrate treatment of HCT-116 colorectal cancer cells. Herein we used quantitative proteomics approaches using iTRAQ (isobaric tags for relative and absolute quantitation), a stable isotope labeling methodology that enables multiplexing of four samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cleavable ICAT, which selectively tags cysteine-containing proteins, was also used, and the results complemented those obtained from the iTRAQ strategy. Selected protein targets were validated by real time PCR and Western blotting. A model is proposed to illustrate our findings from this temporal analysis of the butyrate-responsive proteome that uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer.
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Chu, Trang T T; Sinha, Ameya; Malleret, Benoit; Suwanarusk, Rossarin; Park, Jung E; Naidu, Renugah; Das, Rupambika; Dutta, Bamaprasad; Ong, Seow Theng; Verma, Navin K; Chan, Jerry K; Nosten, François; Rénia, Laurent; Sze, Siu K; Russell, Bruce; Chandramohanadas, Rajesh
2018-01-01
Erythropoiesis is marked by progressive changes in morphological, biochemical and mechanical properties of erythroid precursors to generate red blood cells (RBC). The earliest enucleated forms derived in this process, known as reticulocytes, are multi-lobular and spherical. As reticulocytes mature, they undergo a series of dynamic cytoskeletal re-arrangements and the expulsion of residual organelles, resulting in highly deformable biconcave RBCs (normocytes). To understand the significant, yet neglected proteome-wide changes associated with reticulocyte maturation, we undertook a quantitative proteomics approach. Immature reticulocytes (marked by the presence of surface transferrin receptor, CD71) and mature RBCs (devoid of CD71) were isolated from human cord blood using a magnetic separation procedure. After sub-fractionation into triton-extracted membrane proteins and luminal samples (isobaric tags for relative and absolute quantitation), quantitative mass spectrometry was conducted to identify more than 1800 proteins with good confidence and coverage. While most structural proteins (such as Spectrins, Ankyrin and Band 3) as well as surface glycoproteins were conserved, proteins associated with microtubule structures, such as Talin-1/2 and ß-Tubulin, were detected only in immature reticulocytes. Atomic force microscopy (AFM)-based imaging revealed an extended network of spectrin filaments in reticulocytes (with an average length of 48 nm), which shortened during reticulocyte maturation (average spectrin length of 41 nm in normocytes). The extended nature of cytoskeletal network may partly account for increased deformability and shape changes, as reticulocytes transform to normocytes. © 2017 John Wiley & Sons Ltd.
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Schilling, Birgit; Rardin, Matthew J.; MacLean, Brendan X.; Zawadzka, Anna M.; Frewen, Barbara E.; Cusack, Michael P.; Sorensen, Dylan J.; Bereman, Michael S.; Jing, Enxuan; Wu, Christine C.; Verdin, Eric; Kahn, C. Ronald; MacCoss, Michael J.; Gibson, Bradford W.
2012-01-01
Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models. PMID:22454539
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Hulme, Charlotte H; Wilson, Emma L; Fuller, Heidi R; Roberts, Sally; Richardson, James B; Gallacher, Pete; Peffers, Mandy J; Shirran, Sally L; Botting, Catherine H; Wright, Karina T
2018-05-02
Autologous chondrocyte implantation (ACI) has a failure rate of approximately 20%, but it is yet to be fully understood why. Biomarkers are needed that can pre-operatively predict in which patients it is likely to fail, so that alternative or individualised therapies can be offered. We previously used label-free quantitation (LF) with a dynamic range compression proteomic approach to assess the synovial fluid (SF) of ACI responders and non-responders. However, we were able to identify only a few differentially abundant proteins at baseline. In the present study, we built upon these previous findings by assessing higher-abundance proteins within this SF, providing a more global proteomic analysis on the basis of which more of the biology underlying ACI success or failure can be understood. Isobaric tagging for relative and absolute quantitation (iTRAQ) proteomic analysis was used to assess SF from ACI responders (mean Lysholm improvement of 33; n = 14) and non-responders (mean Lysholm decrease of 14; n = 13) at the two stages of surgery (cartilage harvest and chondrocyte implantation). Differentially abundant proteins in iTRAQ and combined iTRAQ and LF datasets were investigated using pathway and network analyses. iTRAQ proteomic analysis confirmed our previous finding that there is a marked proteomic shift in response to cartilage harvest (70 and 54 proteins demonstrating ≥ 2.0-fold change and p < 0.05 between stages I and II in responders and non-responders, respectively). Further, it highlighted 28 proteins that were differentially abundant between responders and non-responders to ACI, which were not found in the LF study, 16 of which were altered at baseline. The differential expression of two proteins (complement C1s subcomponent and matrix metalloproteinase 3) was confirmed biochemically. Combination of the iTRAQ and LF proteomic datasets generated in-depth SF proteome information that was used to generate interactome networks representing ACI
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Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Applications in Quantitative Proteomics.
Chahrour, Osama; Malone, John
2017-01-01
Recent advances in inductively coupled plasma mass spectrometry (ICP-MS) hyphenated to different separation techniques have promoted it as a valuable tool in protein/peptide quantification. These emerging ICP-MS applications allow absolute quantification by measuring specific elemental responses. One approach quantifies elements already present in the structure of the target peptide (e.g. phosphorus and sulphur) as natural tags. Quantification of these natural tags allows the elucidation of the degree of protein phosphorylation in addition to absolute protein quantification. A separate approach is based on utilising bi-functional labelling substances (those containing ICP-MS detectable elements), that form a covalent chemical bond with the protein thus creating analogs which are detectable by ICP-MS. Based on the previously established stoichiometries of the labelling reagents, quantification can be achieved. This technique is very useful for the design of precise multiplexed quantitation schemes to address the challenges of biomarker screening and discovery. This review discusses the capabilities and different strategies to implement ICP-MS in the field of quantitative proteomics. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
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Klein, Theo; Viner, Rosa I; Overall, Christopher M
2016-10-28
Adaptive immunity is the specialized defence mechanism in vertebrates that evolved to eliminate pathogens. Specialized lymphocytes recognize specific protein epitopes through antigen receptors to mount potent immune responses, many of which are initiated by nuclear factor-kappa B activation and gene transcription. Most, if not all, pathways in adaptive immunity are further regulated by post-translational modification (PTM) of signalling proteins, e.g. phosphorylation, citrullination, ubiquitination and proteolytic processing. The importance of PTMs is reflected by genetic or acquired defects in these pathways that lead to a dysfunctional immune response. Here we discuss the state of the art in targeted proteomics and systems biology approaches to dissect the PTM landscape specifically regarding ubiquitination and proteolysis in B- and T-cell activation. Recent advances have occurred in methods for specific enrichment and targeted quantitation. Together with improved instrument sensitivity, these advances enable the accurate analysis of often rare PTM events that are opaque to conventional proteomics approaches, now rendering in-depth analysis and pathway dissection possible. We discuss published approaches, including as a case study the profiling of the N-terminome of lymphocytes of a rare patient with a genetic defect in the paracaspase protease MALT1, a key regulator protease in antigen-driven signalling, which was manifested by elevated linear ubiquitination.This article is part of the themed issue 'Quantitative mass spectrometry'. © 2016 The Authors.
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PIQMIe: a web server for semi-quantitative proteomics data management and analysis
Kuzniar, Arnold; Kanaar, Roland
2014-01-01
We present the Proteomics Identifications and Quantitations Data Management and Integration Service or PIQMIe that aids in reliable and scalable data management, analysis and visualization of semi-quantitative mass spectrometry based proteomics experiments. PIQMIe readily integrates peptide and (non-redundant) protein identifications and quantitations from multiple experiments with additional biological information on the protein entries, and makes the linked data available in the form of a light-weight relational database, which enables dedicated data analyses (e.g. in R) and user-driven queries. Using the web interface, users are presented with a concise summary of their proteomics experiments in numerical and graphical forms, as well as with a searchable protein grid and interactive visualization tools to aid in the rapid assessment of the experiments and in the identification of proteins of interest. The web server not only provides data access through a web interface but also supports programmatic access through RESTful web service. The web server is available at http://piqmie.semiqprot-emc.cloudlet.sara.nl or http://www.bioinformatics.nl/piqmie. This website is free and open to all users and there is no login requirement. PMID:24861615
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Stable isotope labelling methods in mass spectrometry-based quantitative proteomics.
Chahrour, Osama; Cobice, Diego; Malone, John
2015-09-10
Mass-spectrometry based proteomics has evolved as a promising technology over the last decade and is undergoing a dramatic development in a number of different areas, such as; mass spectrometric instrumentation, peptide identification algorithms and bioinformatic computational data analysis. The improved methodology allows quantitative measurement of relative or absolute protein amounts, which is essential for gaining insights into their functions and dynamics in biological systems. Several different strategies involving stable isotopes label (ICAT, ICPL, IDBEST, iTRAQ, TMT, IPTL, SILAC), label-free statistical assessment approaches (MRM, SWATH) and absolute quantification methods (AQUA) are possible, each having specific strengths and weaknesses. Inductively coupled plasma mass spectrometry (ICP-MS), which is still widely recognised as elemental detector, has recently emerged as a complementary technique to the previous methods. The new application area for ICP-MS is targeting the fast growing field of proteomics related research, allowing absolute protein quantification using suitable elemental based tags. This document describes the different stable isotope labelling methods which incorporate metabolic labelling in live cells, ICP-MS based detection and post-harvest chemical label tagging for protein quantification, in addition to summarising their pros and cons. Copyright © 2015 Elsevier B.V. All rights reserved.
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Song, Jun; Du, Lina; Li, Li; Kalt, Wilhelmina; Palmer, Leslie Campbell; Fillmore, Sherry; Zhang, Ying; Zhang, ZhaoQi; Li, XiHong
2015-06-03
To better understand the regulation of flavonoid and anthocyanin biosynthesis, a targeted quantitative proteomic investigation employing LC-MS with multiple reaction monitoring was conducted on two strawberry cultivars at three ripening stages. This quantitative proteomic workflow was improved through an OFFGEL electrophoresis to fractionate peptides from total protein digests. A total of 154 peptide transitions from 47 peptides covering 21 proteins and isoforms related to anthocyanin biosynthesis were investigated. The normalized protein abundance, which was measured using isotopically-labeled standards, was significantly changed concurrently with increased anthocyanin content and advanced fruit maturity. The protein abundance of phenylalanine ammonia-lyase; anthocyanidin synthase, chalcone isomerase; flavanone 3-hydroxylase; dihydroflavonol 4-reductase, UDP-glucose:flavonoid-3-O-glucosyltransferase, cytochrome c and cytochrome C oxidase subunit 2, was all significantly increased in fruit of more advanced ripeness. An interaction between cultivar and maturity was also shown with respect to chalcone isomerase. The good correlation between protein abundance and anthocyanin content suggested that a metabolic control point may exist for anthocyanin biosynthesis. This research provides insights into the process of anthocyanin formation in strawberry fruit at the level of protein concentration and reveals possible candidates in the regulation of anthocyanin formation during fruit ripening. To gain insight into the molecular mechanisms contributing to flavonoids and anthocyanin biosynthesis and regulation of strawberry fruit during ripening is challenging due to limited molecular biology tools and established hypothesis. Our targeted proteomic approach employing LC-MS/MS analysis and MRM technique to quantify proteins in relation to flavonoids and anthocyanin biosynthesis and regulation in strawberry fruit during fruit ripening is novel. The identification of peptides
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Witzel, Katja; Surabhi, Giridara-Kumar; Jyothsnakumari, Gottimukkala; Sudhakar, Chinta; Matros, Andrea; Mock, Hans-Peter
2007-04-01
This paper describes the application of the recently introduced fluorescence stain Ruthenium(II)-tris-(bathophenanthroline-disulphonate) (RuBP) on a comparative proteome analysis of two phenotypically different barley lines. We carried out an analysis of protein patterns from 2-D gels of the parental lines of the Oregon Wolfe Barley mapping population DOM and REC and stained with either the conventional colloidal Coomassie Brilliant Blue (cCBB) or with the novel RuBP solution. We wished to experimentally verify the usefulness of such a stain in evaluating the complex pattern of a seed proteome, in comparison to the previously used cCBB staining technique. To validate the efficiency of visualization by both stains, we first compared the overall number of detected protein spots. On average, 790 spots were visible by cCBB staining and 1200 spots by RuBP staining. Then, the intensity of a set of spots was assessed, and changes in relative abundance were determined using image analysis software. As expected, staining with RuBP performed better in quantitation in terms of sensitivity and dynamic range. Furthermore, spots from a cultivar-specific region in the protein map were chosen for identification to asses the gain of biological information due to the staining procedure. From this particular region, eight spots were visualized exclusively by RuBP and identification was successful for all spots, proving the ability to identify even very low abundant proteins. Performance in MS analysis was comparable for both protein stains. Proteins were identified by MALDI-TOF MS peptide mass fingerprinting. This approach was not successful for all spots, due to the restricted entry number for barley in the database. Therefore, we subsequently used LC-ESI-Q-TOF MS/MS and de novo sequencing for identification. Because only an insufficient number of proteins from barley is annotated, an EST-based identification strategy was chosen for our experiment. We wished to test whether under
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Mühlhaus, Timo; Weiss, Julia; Hemme, Dorothea; Sommer, Frederik; Schroda, Michael
2011-01-01
Crop-plant-yield safety is jeopardized by temperature stress caused by the global climate change. To take countermeasures by breeding and/or transgenic approaches it is essential to understand the mechanisms underlying plant acclimation to heat stress. To this end proteomics approaches are most promising, as acclimation is largely mediated by proteins. Accordingly, several proteomics studies, mainly based on two-dimensional gel-tandem MS approaches, were conducted in the past. However, results often were inconsistent, presumably attributable to artifacts inherent to the display of complex proteomes via two-dimensional-gels. We describe here a new approach to monitor proteome dynamics in time course experiments. This approach involves full 15N metabolic labeling and mass spectrometry based quantitative shotgun proteomics using a uniform 15N standard over all time points. It comprises a software framework, IOMIQS, that features batch job mediated automated peptide identification by four parallelized search engines, peptide quantification and data assembly for the processing of large numbers of samples. We have applied this approach to monitor proteome dynamics in a heat stress time course using the unicellular green alga Chlamydomonas reinhardtii as model system. We were able to identify 3433 Chlamydomonas proteins, of which 1116 were quantified in at least three of five time points of the time course. Statistical analyses revealed that levels of 38 proteins significantly increased, whereas levels of 206 proteins significantly decreased during heat stress. The increasing proteins comprise 25 (co-)chaperones and 13 proteins involved in chromatin remodeling, signal transduction, apoptosis, photosynthetic light reactions, and yet unknown functions. Proteins decreasing during heat stress were significantly enriched in functional categories that mediate carbon flux from CO2 and external acetate into protein biosynthesis, which also correlated with a rapid, but fully
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Hart-Smith, Gene; Reis, Rodrigo S.; Waterhouse, Peter M.; Wilkins, Marc R.
2017-01-01
Quantitative proteomics strategies – which are playing important roles in the expanding field of plant molecular systems biology – are traditionally designated as either hypothesis driven or non-hypothesis driven. Many of these strategies aim to select individual peptide ions for tandem mass spectrometry (MS/MS), and to do this mixed hypothesis driven and non-hypothesis driven approaches are theoretically simple to implement. In-depth investigations into the efficacies of such approaches have, however, yet to be described. In this study, using combined samples of unlabeled and metabolically 15N-labeled Arabidopsis thaliana proteins, we investigate the mixed use of targeted data acquisition (TDA) and data dependent acquisition (DDA) – referred to as TDA/DDA – to facilitate both hypothesis driven and non-hypothesis driven quantitative data collection in individual LC-MS/MS experiments. To investigate TDA/DDA for hypothesis driven data collection, 7 miRNA target proteins of differing size and abundance were targeted using inclusion lists comprised of 1558 m/z values, using 3 different TDA/DDA experimental designs. In samples in which targeted peptide ions were of particularly low abundance (i.e., predominantly only marginally above mass analyser detection limits), TDA/DDA produced statistically significant increases in the number of targeted peptides identified (230 ± 8 versus 80 ± 3 for DDA; p = 1.1 × 10-3) and quantified (35 ± 3 versus 21 ± 2 for DDA; p = 0.038) per experiment relative to the use of DDA only. These expected improvements in hypothesis driven data collection were observed alongside unexpected improvements in non-hypothesis driven data collection. Untargeted peptide ions with m/z values matching those in inclusion lists were repeatedly identified and quantified across technical replicate TDA/DDA experiments, resulting in significant increases in the percentages of proteins repeatedly quantified in TDA/DDA experiments only relative to DDA
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Proteomic approach to nanotoxicity.
Matysiak, Magdalena; Kapka-Skrzypczak, Lucyna; Brzóska, Kamil; Gutleb, Arno C; Kruszewski, Marcin
2016-03-30
In recent years a large number of engineered nanomaterials (NMs) have been developed with promising technical benefits for consumers and medical appliances. In addition to already known potentially advantageous biological properties (antibiotic, antifungal and antiviral activity) of NMs, many new medical applications of NMs are foreseen, such as drug carriers, contrast agents, radiopharmaceuticals and many others. However, there is increasing concern about potential environmental and health effects due to NMs exposure. An increasing body of evidence suggests that NMs may trigger undesirable hazardous interactions with biological systems with potential to generate harmful effects. In this review we summarized a current state of knowledge on the proteomics approaches to nanotoxicity, including protein corona formation, in vitro and in vivo effects of exposure to NMs on proteome of different classes of organisms, from bacteria and plants to mammals. The effects of NMs on the proteome of environmentally relevant organisms are also described. Despite the benefit that development of nanotechnology may bring to the society, there are still major gaps of knowledge on the influence of nanomaterials on human health and the environment. Thus, it seems necessary to conduct further interdisciplinary research to fill the knowledge gaps in NM toxicity, using more holistic approaches than offered by conventional biological techniques. “OMICS” techniques will certainly help researchers in this field. In this paper we summarized the current stage of knowledge of the effects of nanoparticles on the proteome of different organisms, including those commonly used as an environmentally relevant indicator organisms.
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Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion.
Pan, Yanbo; Cheng, Kai; Mao, Jiawei; Liu, Fangjie; Liu, Jing; Ye, Mingliang; Zou, Hanfa
2014-10-01
Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins.
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A knowledge-based T2-statistic to perform pathway analysis for quantitative proteomic data
Chen, Yi-Hau
2017-01-01
Approaches to identify significant pathways from high-throughput quantitative data have been developed in recent years. Still, the analysis of proteomic data stays difficult because of limited sample size. This limitation also leads to the practice of using a competitive null as common approach; which fundamentally implies genes or proteins as independent units. The independent assumption ignores the associations among biomolecules with similar functions or cellular localization, as well as the interactions among them manifested as changes in expression ratios. Consequently, these methods often underestimate the associations among biomolecules and cause false positives in practice. Some studies incorporate the sample covariance matrix into the calculation to address this issue. However, sample covariance may not be a precise estimation if the sample size is very limited, which is usually the case for the data produced by mass spectrometry. In this study, we introduce a multivariate test under a self-contained null to perform pathway analysis for quantitative proteomic data. The covariance matrix used in the test statistic is constructed by the confidence scores retrieved from the STRING database or the HitPredict database. We also design an integrating procedure to retain pathways of sufficient evidence as a pathway group. The performance of the proposed T2-statistic is demonstrated using five published experimental datasets: the T-cell activation, the cAMP/PKA signaling, the myoblast differentiation, and the effect of dasatinib on the BCR-ABL pathway are proteomic datasets produced by mass spectrometry; and the protective effect of myocilin via the MAPK signaling pathway is a gene expression dataset of limited sample size. Compared with other popular statistics, the proposed T2-statistic yields more accurate descriptions in agreement with the discussion of the original publication. We implemented the T2-statistic into an R package T2GA, which is available at https
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A knowledge-based T2-statistic to perform pathway analysis for quantitative proteomic data.
Lai, En-Yu; Chen, Yi-Hau; Wu, Kun-Pin
2017-06-01
Approaches to identify significant pathways from high-throughput quantitative data have been developed in recent years. Still, the analysis of proteomic data stays difficult because of limited sample size. This limitation also leads to the practice of using a competitive null as common approach; which fundamentally implies genes or proteins as independent units. The independent assumption ignores the associations among biomolecules with similar functions or cellular localization, as well as the interactions among them manifested as changes in expression ratios. Consequently, these methods often underestimate the associations among biomolecules and cause false positives in practice. Some studies incorporate the sample covariance matrix into the calculation to address this issue. However, sample covariance may not be a precise estimation if the sample size is very limited, which is usually the case for the data produced by mass spectrometry. In this study, we introduce a multivariate test under a self-contained null to perform pathway analysis for quantitative proteomic data. The covariance matrix used in the test statistic is constructed by the confidence scores retrieved from the STRING database or the HitPredict database. We also design an integrating procedure to retain pathways of sufficient evidence as a pathway group. The performance of the proposed T2-statistic is demonstrated using five published experimental datasets: the T-cell activation, the cAMP/PKA signaling, the myoblast differentiation, and the effect of dasatinib on the BCR-ABL pathway are proteomic datasets produced by mass spectrometry; and the protective effect of myocilin via the MAPK signaling pathway is a gene expression dataset of limited sample size. Compared with other popular statistics, the proposed T2-statistic yields more accurate descriptions in agreement with the discussion of the original publication. We implemented the T2-statistic into an R package T2GA, which is available at https
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Gu, Liqing; Robinson, Renã A. S.
2016-01-01
Cysteine is a highly reactive amino acid and is subject to a variety of reversible post-translational modifications (PTMs), including nitrosylation, glutathionylation, palmitoylation, as well as formation of sulfenic acid and disulfides. These modifications are not only involved in normal biological activities, such as enzymatic catalysis, redox signaling and cellular homeostasis, but can also be the result of oxidative damage. Especially in aging and neurodegenerative diseases, oxidative stress leads to aberrant cysteine oxidations that affect protein structure and function leading to neurodegeneration as well as other detrimental effects. Methods that can identify cysteine modifications by type, including the site of modification, as well as the relative stoichiometry of the modification can be very helpful for understanding the role of the thiol proteome and redox homeostasis in the context of disease. Cysteine reversible modifications however, are challenging to investigate as they are low abundant, diverse, and labile especially under endogenous conditions. Thanks to the development of redox proteomic approaches, large-scale quantification of cysteine reversible modifications is possible. These approaches cover a range of strategies to enrich, identify, and quantify cysteine reversible modifications from biological samples. This review will focus on nongel-based redox proteomics workflows that give quantitative information about cysteine PTMs and highlight how these strategies have been useful for investigating the redox thiol proteome in aging and neurodegenerative diseases. PMID:27666938
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PIQMIe: a web server for semi-quantitative proteomics data management and analysis.
Kuzniar, Arnold; Kanaar, Roland
2014-07-01
We present the Proteomics Identifications and Quantitations Data Management and Integration Service or PIQMIe that aids in reliable and scalable data management, analysis and visualization of semi-quantitative mass spectrometry based proteomics experiments. PIQMIe readily integrates peptide and (non-redundant) protein identifications and quantitations from multiple experiments with additional biological information on the protein entries, and makes the linked data available in the form of a light-weight relational database, which enables dedicated data analyses (e.g. in R) and user-driven queries. Using the web interface, users are presented with a concise summary of their proteomics experiments in numerical and graphical forms, as well as with a searchable protein grid and interactive visualization tools to aid in the rapid assessment of the experiments and in the identification of proteins of interest. The web server not only provides data access through a web interface but also supports programmatic access through RESTful web service. The web server is available at http://piqmie.semiqprot-emc.cloudlet.sara.nl or http://www.bioinformatics.nl/piqmie. This website is free and open to all users and there is no login requirement. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
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Stable isotope dimethyl labelling for quantitative proteomics and beyond
Hsu, Jue-Liang; Chen, Shu-Hui
2016-01-01
Stable-isotope reductive dimethylation, a cost-effective, simple, robust, reliable and easy-to- multiplex labelling method, is widely applied to quantitative proteomics using liquid chromatography-mass spectrometry. This review focuses on biological applications of stable-isotope dimethyl labelling for a large-scale comparative analysis of protein expression and post-translational modifications based on its unique properties of the labelling chemistry. Some other applications of the labelling method for sample preparation and mass spectrometry-based protein identification and characterization are also summarized. This article is part of the themed issue ‘Quantitative mass spectrometry’. PMID:27644970
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Sardiu, Mihaela E; Gilmore, Joshua M; Carrozza, Michael J; Li, Bing; Workman, Jerry L; Florens, Laurence; Washburn, Michael P
2009-10-06
Protein complexes are key molecular machines executing a variety of essential cellular processes. Despite the availability of genome-wide protein-protein interaction studies, determining the connectivity between proteins within a complex remains a major challenge. Here we demonstrate a method that is able to predict the relationship of proteins within a stable protein complex. We employed a combination of computational approaches and a systematic collection of quantitative proteomics data from wild-type and deletion strain purifications to build a quantitative deletion-interaction network map and subsequently convert the resulting data into an interdependency-interaction model of a complex. We applied this approach to a data set generated from components of the Saccharomyces cerevisiae Rpd3 histone deacetylase complexes, which consists of two distinct small and large complexes that are held together by a module consisting of Rpd3, Sin3 and Ume1. The resulting representation reveals new protein-protein interactions and new submodule relationships, providing novel information for mapping the functional organization of a complex.
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Martyniuk, Christopher J; Popesku, Jason T; Chown, Brittany; Denslow, Nancy D; Trudeau, Vance L
2012-05-01
Neuroendocrine systems integrate both extrinsic and intrinsic signals to regulate virtually all aspects of an animal's physiology. In aquatic toxicology, studies have shown that pollutants are capable of disrupting the neuroendocrine system of teleost fish, and many chemicals found in the environment can also have a neurotoxic mode of action. Omics approaches are now used to better understand cell signaling cascades underlying fish neurophysiology and the control of pituitary hormone release, in addition to identifying adverse effects of pollutants in the teleostean central nervous system. For example, both high throughput genomics and proteomic investigations of molecular signaling cascades for both neurotransmitter and nuclear receptor agonists/antagonists have been reported. This review highlights recent studies that have utilized quantitative proteomics methods such as 2D differential in-gel electrophoresis (DIGE) and isobaric tagging for relative and absolute quantitation (iTRAQ) in neuroendocrine regions and uses these examples to demonstrate the challenges of using proteomics in neuroendocrinology and neurotoxicology research. To begin to characterize the teleost neuroproteome, we functionally annotated 623 unique proteins found in the fish hypothalamus and telencephalon. These proteins have roles in biological processes that include synaptic transmission, ATP production, receptor activity, cell structure and integrity, and stress responses. The biological processes most represented by proteins detected in the teleost neuroendocrine brain included transport (8.4%), metabolic process (5.5%), and glycolysis (4.8%). We provide an example of using sub-network enrichment analysis (SNEA) to identify protein networks in the fish hypothalamus in response to dopamine receptor signaling. Dopamine signaling altered the abundance of proteins that are binding partners of microfilaments, integrins, and intermediate filaments, consistent with data suggesting dopaminergic
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Proteomic approaches in research of cyanobacterial photosynthesis.
Battchikova, Natalia; Angeleri, Martina; Aro, Eva-Mari
2015-10-01
Oxygenic photosynthesis in cyanobacteria, algae, and plants is carried out by a fabulous pigment-protein machinery that is amazingly complicated in structure and function. Many different approaches have been undertaken to characterize the most important aspects of photosynthesis, and proteomics has become the essential component in this research. Here we describe various methods which have been used in proteomic research of cyanobacteria, and demonstrate how proteomics is implemented into on-going studies of photosynthesis in cyanobacterial cells.
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Zanivan, Sara; Maione, Federica; Hein, Marco Y; Hernández-Fernaud, Juan Ramon; Ostasiewicz, Pawel; Giraudo, Enrico; Mann, Matthias
2013-12-01
Proteomics has been successfully used for cell culture on dishes, but more complex cellular systems have proven to be challenging and so far poorly approached with proteomics. Because of the complexity of the angiogenic program, we still do not have a complete understanding of the molecular mechanisms involved in this process, and there have been no in depth quantitative proteomic studies. Plating endothelial cells on matrigel recapitulates aspects of vessel growth, and here we investigate this mechanism by using a spike-in SILAC quantitative proteomic approach. By comparing proteomic changes in primary human endothelial cells morphogenesis on matrigel to general adhesion mechanisms in cells spreading on culture dish, we pinpoint pathways and proteins modulated by endothelial cells. The cell-extracellular matrix adhesion proteome depends on the adhesion substrate, and a detailed proteomic profile of the extracellular matrix secreted by endothelial cells identified CLEC14A as a matrix component, which binds to MMRN2. We verify deregulated levels of these proteins during tumor angiogenesis in models of multistage carcinogenesis. This is the most in depth quantitative proteomic study of endothelial cell morphogenesis, which shows the potential of applying high accuracy quantitative proteomics to in vitro models of vessel growth to shed new light on mechanisms that accompany pathological angiogenesis. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000359.
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Quantitative Proteomic Analysis of the Hfq-Regulon in Sinorhizobium meliloti 2011
Sobrero, Patricio; Schlüter, Jan-Philip; Lanner, Ulrike; Schlosser, Andreas; Becker, Anke; Valverde, Claudio
2012-01-01
Riboregulation stands for RNA-based control of gene expression. In bacteria, small non-coding RNAs (sRNAs) are a major class of riboregulatory elements, most of which act at the post-transcriptional level by base-pairing target mRNA genes. The RNA chaperone Hfq facilitates antisense interactions between target mRNAs and regulatory sRNAs, thus influencing mRNA stability and/or translation rate. In the α-proteobacterium Sinorhizobium meliloti strain 2011, the identification and detection of multiple sRNAs genes and the broadly pleitropic phenotype associated to the absence of a functional Hfq protein both support the existence of riboregulatory circuits controlling gene expression to ensure the fitness of this bacterium in both free living and symbiotic conditions. In order to identify target mRNAs subject to Hfq-dependent riboregulation, we have compared the proteome of an hfq mutant and the wild type S. meliloti by quantitative proteomics following protein labelling with 15N. Among 2139 univocally identified proteins, a total of 195 proteins showed a differential abundance between the Hfq mutant and the wild type strain; 65 proteins accumulated ≥2-fold whereas 130 were downregulated (≤0.5-fold) in the absence of Hfq. This profound proteomic impact implies a major role for Hfq on regulation of diverse physiological processes in S. meliloti, from transport of small molecules to homeostasis of iron and nitrogen. Changes in the cellular levels of proteins involved in transport of nucleotides, peptides and amino acids, and in iron homeostasis, were confirmed with phenotypic assays. These results represent the first quantitative proteomic analysis in S. meliloti. The comparative analysis of the hfq mutant proteome allowed identification of novel strongly Hfq-regulated genes in S. meliloti. PMID:23119037
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Quantitative proteomic analysis of the Hfq-regulon in Sinorhizobium meliloti 2011.
Sobrero, Patricio; Schlüter, Jan-Philip; Lanner, Ulrike; Schlosser, Andreas; Becker, Anke; Valverde, Claudio
2012-01-01
Riboregulation stands for RNA-based control of gene expression. In bacteria, small non-coding RNAs (sRNAs) are a major class of riboregulatory elements, most of which act at the post-transcriptional level by base-pairing target mRNA genes. The RNA chaperone Hfq facilitates antisense interactions between target mRNAs and regulatory sRNAs, thus influencing mRNA stability and/or translation rate. In the α-proteobacterium Sinorhizobium meliloti strain 2011, the identification and detection of multiple sRNAs genes and the broadly pleitropic phenotype associated to the absence of a functional Hfq protein both support the existence of riboregulatory circuits controlling gene expression to ensure the fitness of this bacterium in both free living and symbiotic conditions. In order to identify target mRNAs subject to Hfq-dependent riboregulation, we have compared the proteome of an hfq mutant and the wild type S. meliloti by quantitative proteomics following protein labelling with (15)N. Among 2139 univocally identified proteins, a total of 195 proteins showed a differential abundance between the Hfq mutant and the wild type strain; 65 proteins accumulated ≥2-fold whereas 130 were downregulated (≤0.5-fold) in the absence of Hfq. This profound proteomic impact implies a major role for Hfq on regulation of diverse physiological processes in S. meliloti, from transport of small molecules to homeostasis of iron and nitrogen. Changes in the cellular levels of proteins involved in transport of nucleotides, peptides and amino acids, and in iron homeostasis, were confirmed with phenotypic assays. These results represent the first quantitative proteomic analysis in S. meliloti. The comparative analysis of the hfq mutant proteome allowed identification of novel strongly Hfq-regulated genes in S. meliloti.
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Stachowicz, Aneta; Siudut, Jakub; Suski, Maciej; Olszanecki, Rafał; Korbut, Ryszard; Undas, Anetta; Wiśniewski, Jacek R
2017-01-01
It is well known that fibrin network binds a large variety of proteins, including inhibitors and activators of fibrinolysis, which may affect clot properties, such as stability and susceptibility to fibrinolysis. Specific plasma clot composition differs between individuals and may change in disease states. However, the plasma clot proteome has not yet been in-depth analyzed, mainly due to technical difficulty related to the presence of a highly abundant protein-fibrinogen and fibrin that forms a plasma clot. The aim of our study was to optimize quantitative proteomic analysis of fibrin clots prepared ex vivo from citrated plasma of the peripheral blood drawn from patients with prior venous thromboembolism (VTE). We used a multiple enzyme digestion filter aided sample preparation, a multienzyme digestion (MED) FASP method combined with LC-MS/MS analysis performed on a Proxeon Easy-nLC System coupled to the Q Exactive HF mass spectrometer. We also evaluated the impact of peptide fractionation with pipet-tip strong anion exchange (SAX) method on the obtained results. Our proteomic approach revealed 476 proteins repeatedly identified in the plasma fibrin clots from patients with VTE including extracellular vesicle-derived proteins, lipoproteins, fibrinolysis inhibitors, and proteins involved in immune responses. The MED FASP method using three different enzymes: LysC, trypsin and chymotrypsin increased the number of identified peptides and proteins and their sequence coverage as compared to a single step digestion. Peptide fractionation with a pipet-tip strong anion exchange (SAX) protocol increased the depth of proteomic analyses, but also extended the time needed for sample analysis with LC-MS/MS. The MED FASP method combined with a label-free quantification is an excellent proteomic approach for the analysis of fibrin clots prepared ex vivo from citrated plasma of patients with prior VTE.
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Magnetoresistive biosensors for quantitative proteomics
NASA Astrophysics Data System (ADS)
Zhou, Xiahan; Huang, Chih-Cheng; Hall, Drew A.
2017-08-01
Quantitative proteomics, as a developing method for study of proteins and identification of diseases, reveals more comprehensive and accurate information of an organism than traditional genomics. A variety of platforms, such as mass spectrometry, optical sensors, electrochemical sensors, magnetic sensors, etc., have been developed for detecting proteins quantitatively. The sandwich immunoassay is widely used as a labeled detection method due to its high specificity and flexibility allowing multiple different types of labels. While optical sensors use enzyme and fluorophore labels to detect proteins with high sensitivity, they often suffer from high background signal and challenges in miniaturization. Magnetic biosensors, including nuclear magnetic resonance sensors, oscillator-based sensors, Hall-effect sensors, and magnetoresistive sensors, use the specific binding events between magnetic nanoparticles (MNPs) and target proteins to measure the analyte concentration. Compared with other biosensing techniques, magnetic sensors take advantage of the intrinsic lack of magnetic signatures in biological samples to achieve high sensitivity and high specificity, and are compatible with semiconductor-based fabrication process to have low-cost and small-size for point-of-care (POC) applications. Although still in the development stage, magnetic biosensing is a promising technique for in-home testing and portable disease monitoring.
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An Overview of Advanced SILAC-Labeling Strategies for Quantitative Proteomics.
Terzi, F; Cambridge, S
2017-01-01
Comparative, quantitative mass spectrometry of proteins provides great insight to protein abundance and function, but some molecular characteristics related to protein dynamics are not so easily obtained. Because the metabolic incorporation of stable amino acid isotopes allows the extraction of distinct temporal and spatial aspects of protein dynamics, the SILAC methodology is uniquely suited to be adapted for advanced labeling strategies. New SILAC strategies have emerged that allow deeper foraging into the complexity of cellular proteomes. Here, we review a few advanced SILAC-labeling strategies that have been published during last the years. Among them, different subsaturating-labeling as well as dual-labeling schemes are most prominent for a range of analyses including those of neuronal proteomes, secretion, or cell-cell-induced stimulations. These recent developments suggest that much more information can be gained from proteomic analyses if the labeling strategies are specifically tailored toward the experimental design. © 2017 Elsevier Inc. All rights reserved.
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Yang, Yongxin; Bu, Dengpan; Zhao, Xiaowei; Sun, Peng; Wang, Jiaqi; Zhou, Lingyun
2013-04-05
To aid in unraveling diverse genetic and biological unknowns, a proteomic approach was used to analyze the whey proteome in cow, yak, buffalo, goat, and camel milk based on the isobaric tag for relative and absolute quantification (iTRAQ) techniques. This analysis is the first to produce proteomic data for the milk from the above-mentioned animal species: 211 proteins have been identified and 113 proteins have been categorized according to molecular function, cellular components, and biological processes based on gene ontology annotation. The results of principal component analysis showed significant differences in proteomic patterns among goat, camel, cow, buffalo, and yak milk. Furthermore, 177 differentially expressed proteins were submitted to advanced hierarchical clustering. The resulting clustering pattern included three major sample clusters: (1) cow, buffalo, and yak milk; (2) goat, cow, buffalo, and yak milk; and (3) camel milk. Certain proteins were chosen as characterization traits for a given species: whey acidic protein and quinone oxidoreductase for camel milk, biglycan for goat milk, uncharacterized protein (Accession Number: F1MK50 ) for yak milk, clusterin for buffalo milk, and primary amine oxidase for cow milk. These results help reveal the quantitative milk whey proteome pattern for analyzed species. This provides information for evaluating adulteration of specific specie milk and may provide potential directions for application of specific milk protein production based on physiological differences among animal species.
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Alvarez, Sophie; Roy Choudhury, Swarup; Hicks, Leslie M; Pandey, Sona
2013-03-01
Abscisic acid (ABA) is proposed to be perceived by multiple receptors in plants. We have previously reported on the role of two GPCR-type G-proteins (GTG proteins) as plasma membrane-localized ABA receptors in Arabidopsis thaliana. However, due to the presence of multiple transmembrane domains, detailed structural and biochemical characterization of GTG proteins remains limited. Since ABA induces substantial changes in the proteome of plants, a labeling LC-based quantitative proteomics approach was applied to elucidate the global effects and possible downstream targets of GTG1/GTG2 proteins. Quantitative differences in protein abundance between wild-type and gtg1gtg2 were analyzed for evaluation of the effect of ABA on the root proteome and its dependence on the presence of functional GTG1/GTG2 proteins. The results presented in this study reveal the most comprehensive ABA-responsive root proteome reported to date in Arabidopsis. Notably, the majority of ABA-responsive proteins required the presence of GTG proteins, supporting their key role in ABA signaling. These observations were further confirmed by additional experiments. Overall, comparison of the ABA-dependent protein abundance changes in wild-type versus gtg1gtg2 provides clues to their possible links with some of the well-established effectors of the ABA signaling pathways and their role in mediating phytohormone cross-talk.
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Morris, Jeffrey S; Baladandayuthapani, Veerabhadran; Herrick, Richard C; Sanna, Pietro; Gutstein, Howard
2011-01-01
Image data are increasingly encountered and are of growing importance in many areas of science. Much of these data are quantitative image data, which are characterized by intensities that represent some measurement of interest in the scanned images. The data typically consist of multiple images on the same domain and the goal of the research is to combine the quantitative information across images to make inference about populations or interventions. In this paper, we present a unified analysis framework for the analysis of quantitative image data using a Bayesian functional mixed model approach. This framework is flexible enough to handle complex, irregular images with many local features, and can model the simultaneous effects of multiple factors on the image intensities and account for the correlation between images induced by the design. We introduce a general isomorphic modeling approach to fitting the functional mixed model, of which the wavelet-based functional mixed model is one special case. With suitable modeling choices, this approach leads to efficient calculations and can result in flexible modeling and adaptive smoothing of the salient features in the data. The proposed method has the following advantages: it can be run automatically, it produces inferential plots indicating which regions of the image are associated with each factor, it simultaneously considers the practical and statistical significance of findings, and it controls the false discovery rate. Although the method we present is general and can be applied to quantitative image data from any application, in this paper we focus on image-based proteomic data. We apply our method to an animal study investigating the effects of opiate addiction on the brain proteome. Our image-based functional mixed model approach finds results that are missed with conventional spot-based analysis approaches. In particular, we find that the significant regions of the image identified by the proposed method
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Quantitative proteomic analysis of bacterial enzymes released in cheese during ripening.
Jardin, Julien; Mollé, Daniel; Piot, Michel; Lortal, Sylvie; Gagnaire, Valérie
2012-04-02
Due to increasingly available bacterial genomes in databases, proteomic tools have recently been used to screen proteins expressed by micro-organisms in food in order to better understand their metabolism in situ. While the main objective is the systematic identification of proteins, the next step will be to bridge the gap between identification and quantification of these proteins. For that purpose, a new mass spectrometry-based approach was applied, using isobaric tagging reagent for quantitative proteomic analysis (iTRAQ), which are amine specific and yield labelled peptides identical in mass. Experimental Swiss-type cheeses were manufactured from microfiltered milk using Streptococcus thermophilus ITG ST20 and Lactobacillus helveticus ITG LH1 as lactic acid starters. At three ripening times (7, 20 and 69 days), cheese aqueous phases were extracted and enriched in bacterial proteins by fractionation. Each sample, standardised in protein amount prior to proteomic analyses, was: i) analysed by 2D-electrophoresis for qualitative analysis and ii) submitted to trypsinolysis, and labelled with specific iTRAQ tag, one per ripening time. The three labelled samples were mixed together and analysed by nano-LC coupled on-line with ESI-QTOF mass spectrometer. Thirty proteins, both from bacterial or bovine origin, were identified and efficiently quantified. The free bacterial proteins detected were enzymes from the central carbon metabolism as well as stress proteins. Depending on the protein considered, the quantity of these proteins in the cheese aqueous extract increased from 2.5 to 20 fold in concentration from day 7 to day 69 of ripening. Copyright © 2012 Elsevier B.V. All rights reserved.
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López-Ferrer, Daniel; Hixson, Kim K.; Smallwood, Heather; Squier, Thomas C.; Petritis, Konstantinos; Smith, Richard D.
2009-01-01
A new method that uses immobilized trypsin concomitant with ultrasonic irradiation results in ultra-rapid digestion and thorough 18O labeling for quantitative protein comparisons. The reproducible and highly efficient method provided effective digestions in <1 min with a minimized amount of enzyme required compared to traditional methods. This method was demonstrated for digestion of both simple and complex protein mixtures, including bovine serum albumin, a global proteome extract from the bacteria Shewanella oneidensis, and mouse plasma, as well as 18O labeling of such complex protein mixtures, which validated the application of this method for differential proteomic measurements. This approach is simple, reproducible, cost effective, rapid, and thus well-suited for automation. PMID:19555078
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A Quantitative Spatial Proteomics Analysis of Proteome Turnover in Human Cells*
Boisvert, François-Michel; Ahmad, Yasmeen; Gierliński, Marek; Charrière, Fabien; Lamont, Douglas; Scott, Michelle; Barton, Geoff; Lamond, Angus I.
2012-01-01
Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ∼20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general
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Hempel, Kristina; Herbst, Florian-Alexander; Moche, Martin; Hecker, Michael; Becher, Dörte
2011-04-01
Staphylococcus aureus is capable of colonizing and infecting humans by its arsenal of surface-exposed and secreted proteins. Iron-limited conditions in mammalian body fluids serve as a major environmental signal to bacteria to express virulence determinants. Here we present a comprehensive, gel-free, and GeLC-MS/MS-based quantitative proteome profiling of S. aureus under this infection-relevant situation. (14)N(15)N metabolic labeling and three complementing approaches were combined for relative quantitative analyses of surface-associated proteins. The surface-exposed and secreted proteome profiling approaches comprise trypsin shaving, biotinylation, and precipitation of the supernatant. By analysis of the outer subproteomic and cytoplasmic protein fraction, 1210 proteins could be identified including 221 surface-associated proteins. Thus, access was enabled to 70% of the predicted cell wall-associated proteins, 80% of the predicted sortase substrates, two/thirds of lipoproteins and more than 50% of secreted and cytoplasmic proteins. For iron-deficiency, 158 surface-associated proteins were quantified. Twenty-nine proteins were found in altered amounts showing particularly surface-exposed proteins strongly induced, such as the iron-regulated surface determinant proteins IsdA, IsdB, IsdC and IsdD as well as lipid-anchored iron compound-binding proteins. The work presents a crucial subject for understanding S. aureus pathophysiology by the use of methods that allow quantitative surface proteome profiling.
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Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan
2016-04-13
Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish.
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Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan
2016-01-01
Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish. PMID:27071722
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Ou, Keli; Kesuma, Djohan; Ganesan, Kumaresan; Yu, Kun; Soon, Sou Yen; Lee, Suet Ying; Goh, Xin Pei; Hooi, Michelle; Chen, Wei; Jikuya, Hiroyuki; Ichikawa, Tetsuo; Kuyama, Hiroki; Matsuo, Ei-ichi; Nishimura, Osamu; Tan, Patrick
2006-09-01
The identification of drug-responsive biomarkers in complex protein mixtures is an important goal of quantitative proteomics. Here, we describe a novel approach for identifying such drug-induced protein alterations, which combines 2-nitrobenzenesulfenyl chloride (NBS) tryptophan labeling with two-dimensional gel electrophoresis (2DE)/mass spectrometry (MS). Lysates from drug-treated and control samples are labeled with light or heavy NBS moiety and separated on a common 2DE gel, and protein alterations are identified by MS through the differential intensity of paired NBS peptide peaks. Using NBS/2DE/MS, we profiled the proteomic alterations induced by tamoxifen (TAM) in the estrogen receptor (ER) positive MCF-7 breast cancer cell line. Of 88 protein spots that significantly changed upon TAM treatment, 44 spots representing 23 distinct protein species were successfully identified with NBS-paired peptides. Of these 23 TAM-altered proteins, 16 (70%) have not been previously associated with TAM or ER activity. We found the NBS labeling procedure to be both technically and biologically reproducible, and the NBS/2DE/MS alterations exhibited good concordance with conventional 2DE differential protein quantitation, with discrepancies largely due to the comigration of distinct proteins in the regular 2DE gels. To validate the NBS/2DE/MS results, we used immunoblotting to confirm GRP78, CK19, and PA2G4 as bona fide TAM-regulated proteins. Furthermore, we demonstrate that PA2G4 expression can serve as a novel prognostic factor for disease-free survival in two independent breast cancer patient cohorts. To our knowledge, this is the first report describing the proteomic changes in breast cancer cells induced by TAM, the most commonly used selective estrogen receptor modulator (SERM). Our results indicate that NBS/2DE/MS may represent a more reliable approach for cellular protein quantitation than conventional 2DE approaches.
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Wiśniewski, Jacek R; Mann, Matthias
2016-07-01
Proteomics and other protein-based analysis methods such as Western blotting all face the challenge of discriminating changes in the levels of proteins of interest from inadvertent changes in the amount loaded for analysis. Mass-spectrometry-based proteomics can now estimate the relative and absolute amounts of thousands of proteins across diverse biological systems. We reasoned that this new technology could prove useful for selection of very stably expressed proteins that could serve as better loading controls than those traditionally employed. Large-scale proteomic analyses of SDS lysates of cultured cells and tissues revealed deglycase DJ-1 as the protein with the lowest variability in abundance among different cell types in human, mouse, and amphibian cells. The protein constitutes 0.069 ± 0.017% of total cellular protein and occurs at a specific concentration of 34.6 ± 8.7 pmol/mg of total protein. Since DJ-1 is ubiquitous and therefore easily detectable with several peptides, it can be helpful in normalization of proteomic data sets. In addition, DJ-1 appears to be an advantageous loading control for Western blot that is superior to those used commonly used, allowing comparisons between tissues and cells originating from evolutionarily distant vertebrate species. Notably, this is not possible by the detection and quantitation of housekeeping proteins, which are often used in the Western blot technique. The approach introduced here can be applied to select the most appropriate loading controls for MS-based proteomics or Western blotting in any biological system.
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A proteomic approach to identify endosomal cargoes controlling cancer invasiveness
Diaz-Vera, Jesica; Palmer, Sarah; Hernandez-Fernaud, Juan Ramon; Dornier, Emmanuel; Mitchell, Louise E.; Macpherson, Iain; Edwards, Joanne; Zanivan, Sara
2017-01-01
ABSTRACT We have previously shown that Rab17, a small GTPase associated with epithelial polarity, is specifically suppressed by ERK2 (also known as MAPK1) signalling to promote an invasive phenotype. However, the mechanisms through which Rab17 loss permits invasiveness, and the endosomal cargoes that are responsible for mediating this, are unknown. Using quantitative mass spectrometry-based proteomics, we have found that knockdown of Rab17 leads to a highly selective reduction in the cellular levels of a v-SNARE (Vamp8). Moreover, proteomics and immunofluorescence indicate that Vamp8 is associated with Rab17 at late endosomes. Reduced levels of Vamp8 promote transition between ductal carcinoma in situ (DCIS) and a more invasive phenotype. We developed an unbiased proteomic approach to elucidate the complement of receptors that redistributes between endosomes and the plasma membrane, and have pin-pointed neuropilin-2 (NRP2) as a key pro-invasive cargo of Rab17- and Vamp8-regulated trafficking. Indeed, reduced Rab17 or Vamp8 levels lead to increased mobilisation of NRP2-containing late endosomes and upregulated cell surface expression of NRP2. Finally, we show that NRP2 is required for the basement membrane disruption that accompanies the transition between DCIS and a more invasive phenotype. PMID:28062852
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Åhrman, Emma; Hallgren, Oskar; Malmström, Lars; Hedström, Ulf; Malmström, Anders; Bjermer, Leif; Zhou, Xiao-Hong; Westergren-Thorsson, Gunilla; Malmström, Johan
2018-03-01
Remodeling of the extracellular matrix (ECM) is a common feature in lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Here, we applied a sequential tissue extraction strategy to describe disease-specific remodeling of human lung tissue in disease, using end-stages of COPD and IPF. Our strategy was based on quantitative comparison of the disease proteomes, with specific focus on the matrisome, using data-independent acquisition and targeted data analysis (SWATH-MS). Our work provides an in-depth proteomic characterization of human lung tissue during impaired tissue remodeling. In addition, we show important quantitative and qualitative effects of the solubility of matrisome proteins. COPD was characterized by a disease-specific increase in ECM regulators, metalloproteinase inhibitor 3 (TIMP3) and matrix metalloproteinase 28 (MMP-28), whereas for IPF, impairment in cell adhesion proteins, such as collagen VI and laminins, was most prominent. For both diseases, we identified increased levels of proteins involved in the regulation of endopeptidase activity, with several proteins belonging to the serpin family. The established human lung quantitative proteome inventory and the construction of a tissue-specific protein assay library provides a resource for future quantitative proteomic analyses of human lung tissues. We present a sequential tissue extraction strategy to determine changes in extractability of matrisome proteins in end-stage COPD and IPF compared to healthy control tissue. Extensive quantitative analysis of the proteome changes of the disease states revealed altered solubility of matrisome proteins involved in ECM regulators and cell-ECM communication. The results highlight disease-specific remodeling mechanisms associated with COPD and IPF. Copyright © 2018 Elsevier B.V. All rights reserved.
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Lo, Andy; Weiner, Joel H; Li, Liang
2013-09-17
Due to limited sample amounts, instrument time considerations, and reagent costs, only a small number of replicate experiments are typically performed for quantitative proteome analyses. Generation of reproducible data that can be readily assessed for consistency within a small number of datasets is critical for accurate quantification. We report our investigation of a strategy using reciprocal isotope labeling of two comparative samples as a tool for determining proteome changes. Reciprocal labeling was evaluated to determine the internal consistency of quantified proteome changes from Escherichia coli grown under aerobic and anaerobic conditions. Qualitatively, the peptide overlap between replicate analyses of the same sample and reverse labeled samples were found to be within 8%. Quantitatively, reciprocal analyses showed only a slight increase in average overall inconsistency when compared with replicate analyses (1.29 vs. 1.24-fold difference). Most importantly, reverse labeling was successfully used to identify spurious values resulting from incorrect peptide identifications and poor peak fitting. After removal of 5% of the peptide data with low reproducibility, a total of 275 differentially expressed proteins (>1.50-fold difference) were consistently identified and were then subjected to bioinformatics analysis. General considerations and guidelines for reciprocal labeling experimental design and biological significance of obtained results are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.
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Goeminne, Ludger J E; Gevaert, Kris; Clement, Lieven
2018-01-16
Label-free shotgun proteomics is routinely used to assess proteomes. However, extracting relevant information from the massive amounts of generated data remains difficult. This tutorial provides a strong foundation on analysis of quantitative proteomics data. We provide key statistical concepts that help researchers to design proteomics experiments and we showcase how to analyze quantitative proteomics data using our recent free and open-source R package MSqRob, which was developed to implement the peptide-level robust ridge regression method for relative protein quantification described by Goeminne et al. MSqRob can handle virtually any experimental proteomics design and outputs proteins ordered by statistical significance. Moreover, its graphical user interface and interactive diagnostic plots provide easy inspection and also detection of anomalies in the data and flaws in the data analysis, allowing deeper assessment of the validity of results and a critical review of the experimental design. Our tutorial discusses interactive preprocessing, data analysis and visualization of label-free MS-based quantitative proteomics experiments with simple and more complex designs. We provide well-documented scripts to run analyses in bash mode on GitHub, enabling the integration of MSqRob in automated pipelines on cluster environments (https://github.com/statOmics/MSqRob). The concepts outlined in this tutorial aid in designing better experiments and analyzing the resulting data more appropriately. The two case studies using the MSqRob graphical user interface will contribute to a wider adaptation of advanced peptide-based models, resulting in higher quality data analysis workflows and more reproducible results in the proteomics community. We also provide well-documented scripts for experienced users that aim at automating MSqRob on cluster environments. Copyright © 2017 Elsevier B.V. All rights reserved.
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Quantitative Proteomics via High Resolution MS Quantification: Capabilities and Limitations
Higgs, Richard E.; Butler, Jon P.; Han, Bomie; Knierman, Michael D.
2013-01-01
Recent improvements in the mass accuracy and resolution of mass spectrometers have led to renewed interest in label-free quantification using data from the primary mass spectrum (MS1) acquired from data-dependent proteomics experiments. The capacity for higher specificity quantification of peptides from samples enriched for proteins of biological interest offers distinct advantages for hypothesis generating experiments relative to immunoassay detection methods or prespecified peptide ions measured by multiple reaction monitoring (MRM) approaches. Here we describe an evaluation of different methods to post-process peptide level quantification information to support protein level inference. We characterize the methods by examining their ability to recover a known dilution of a standard protein in background matrices of varying complexity. Additionally, the MS1 quantification results are compared to a standard, targeted, MRM approach on the same samples under equivalent instrument conditions. We show the existence of multiple peptides with MS1 quantification sensitivity similar to the best MRM peptides for each of the background matrices studied. Based on these results we provide recommendations on preferred approaches to leveraging quantitative measurements of multiple peptides to improve protein level inference. PMID:23710359
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Lehmann, Sylvain; Hoofnagle, Andrew; Hochstrasser, Denis; Brede, Cato; Glueckmann, Matthias; Cocho, José A; Ceglarek, Uta; Lenz, Christof; Vialaret, Jérôme; Scherl, Alexander; Hirtz, Christophe
2013-05-01
Proteomics studies typically aim to exhaustively detect peptides/proteins in a given biological sample. Over the past decade, the number of publications using proteomics methodologies has exploded. This was made possible due to the availability of high-quality genomic data and many technological advances in the fields of microfluidics and mass spectrometry. Proteomics in biomedical research was initially used in 'functional' studies for the identification of proteins involved in pathophysiological processes, complexes and networks. Improved sensitivity of instrumentation facilitated the analysis of even more complex sample types, including human biological fluids. It is at that point the field of clinical proteomics was born, and its fundamental aim was the discovery and (ideally) validation of biomarkers for the diagnosis, prognosis, or therapeutic monitoring of disease. Eventually, it was recognized that the technologies used in clinical proteomics studies [particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS)] could represent an alternative to classical immunochemical assays. Prior to deploying MS in the measurement of peptides/proteins in the clinical laboratory, it seems likely that traditional proteomics workflows and data management systems will need to adapt to the clinical environment and meet in vitro diagnostic (IVD) regulatory constraints. This defines a new field, as reviewed in this article, that we have termed quantitative Clinical Chemistry Proteomics (qCCP).
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A tutorial for software development in quantitative proteomics using PSI standard formats☆
Gonzalez-Galarza, Faviel F.; Qi, Da; Fan, Jun; Bessant, Conrad; Jones, Andrew R.
2014-01-01
The Human Proteome Organisation — Proteomics Standards Initiative (HUPO-PSI) has been working for ten years on the development of standardised formats that facilitate data sharing and public database deposition. In this article, we review three HUPO-PSI data standards — mzML, mzIdentML and mzQuantML, which can be used to design a complete quantitative analysis pipeline in mass spectrometry (MS)-based proteomics. In this tutorial, we briefly describe the content of each data model, sufficient for bioinformaticians to devise proteomics software. We also provide guidance on the use of recently released application programming interfaces (APIs) developed in Java for each of these standards, which makes it straightforward to read and write files of any size. We have produced a set of example Java classes and a basic graphical user interface to demonstrate how to use the most important parts of the PSI standards, available from http://code.google.com/p/psi-standard-formats-tutorial. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan. PMID:23584085
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Vester, Diana; Rapp, Erdmann; Gade, Dörte; Genzel, Yvonne; Reichl, Udo
2009-06-01
Over the last years virus-host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2-D DIGE and nanoHPLC-nanoESI-MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2-D gels of the proteomes of uninfected and influenza-infected host cells, 16 quantitatively altered protein spots (at least +/-1.7-fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon-induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome-wide profiling of virus infection can provide insights into complexity and dynamics of virus-host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.
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Wu, Yi-Hsuan; Hu, Chia-Wei; Chien, Chih-Wei; Chen, Yu-Ju; Huang, Hsuan-Cheng; Juan, Hsueh-Fen
2013-01-01
ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.
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Wu, Yi-Hsuan; Hu, Chia-Wei; Chien, Chih-Wei; Chen, Yu-Ju; Huang, Hsuan-Cheng; Juan, Hsueh-Fen
2013-01-01
ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy. PMID:23990911
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Quantitative proteome analysis using isobaric peptide termini labeling (IPTL).
Arntzen, Magnus O; Koehler, Christian J; Treumann, Achim; Thiede, Bernd
2011-01-01
The quantitative comparison of proteome level changes across biological samples has become an essential feature in proteomics that remains challenging. We have recently introduced isobaric peptide termini labeling (IPTL), a novel strategy for isobaric quantification based on the derivatization of peptide termini with complementary isotopically labeled reagents. Unlike non-isobaric quantification methods, sample complexity at the MS level is not increased, providing improved sensitivity and protein coverage. The distinguishing feature of IPTL when comparing it to more established isobaric labeling methods (iTRAQ and TMT) is the presence of quantification signatures in all sequence-determining ions in MS/MS spectra, not only in the low mass reporter ion region. This makes IPTL a quantification method that is accessible to mass spectrometers with limited capabilities in the low mass range. Also, the presence of several quantification points in each MS/MS spectrum increases the robustness of the quantification procedure.
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A statistical framework for protein quantitation in bottom-up MS-based proteomics
DOE Office of Scientific and Technical Information (OSTI.GOV)
Karpievitch, Yuliya; Stanley, Jeffrey R.; Taverner, Thomas
2009-08-15
ABSTRACT Motivation: Quantitative mass spectrometry-based proteomics requires protein-level estimates and confidence measures. Challenges include the presence of low-quality or incorrectly identified peptides and widespread, informative, missing data. Furthermore, models are required for rolling peptide-level information up to the protein level. Results: We present a statistical model for protein abundance in terms of peptide peak intensities, applicable to both label-based and label-free quantitation experiments. The model allows for both random and censoring missingness mechanisms and provides naturally for protein-level estimates and confidence measures. The model is also used to derive automated filtering and imputation routines. Three LC-MS datasets are used tomore » illustrate the methods. Availability: The software has been made available in the open-source proteomics platform DAnTE (Polpitiya et al. (2008)) (http://omics.pnl.gov/software/). Contact: adabney@stat.tamu.edu« less
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Wu, Qi; Yuan, Huiming; Zhang, Lihua; Zhang, Yukui
2012-06-20
With the acceleration of proteome research, increasing attention has been paid to multidimensional liquid chromatography-mass spectrometry (MDLC-MS) due to its high peak capacity and separation efficiency. Recently, many efforts have been put to improve MDLC-based strategies including "top-down" and "bottom-up" to enable highly sensitive qualitative and quantitative analysis of proteins, as well as accelerate the whole analytical procedure. Integrated platforms with combination of sample pretreatment, multidimensional separations and identification were also developed to achieve high throughput and sensitive detection of proteomes, facilitating highly accurate and reproducible quantification. This review summarized the recent advances of such techniques and their applications in qualitative and quantitative analysis of proteomes. Copyright © 2012 Elsevier B.V. All rights reserved.
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A Statistical Framework for Protein Quantitation in Bottom-Up MS-Based Proteomics
DOE Office of Scientific and Technical Information (OSTI.GOV)
Karpievitch, Yuliya; Stanley, Jeffrey R.; Taverner, Thomas
2009-08-15
Motivation: Quantitative mass spectrometry-based proteomics requires protein-level estimates and associated confidence measures. Challenges include the presence of low quality or incorrectly identified peptides and informative missingness. Furthermore, models are required for rolling peptide-level information up to the protein level. Results: We present a statistical model that carefully accounts for informative missingness in peak intensities and allows unbiased, model-based, protein-level estimation and inference. The model is applicable to both label-based and label-free quantitation experiments. We also provide automated, model-based, algorithms for filtering of proteins and peptides as well as imputation of missing values. Two LC/MS datasets are used to illustrate themore » methods. In simulation studies, our methods are shown to achieve substantially more discoveries than standard alternatives. Availability: The software has been made available in the opensource proteomics platform DAnTE (http://omics.pnl.gov/software/). Contact: adabney@stat.tamu.edu Supplementary information: Supplementary data are available at Bioinformatics online.« less
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Zhang, Fanglin; Lin, Hechun; Gu, Aiqin; Li, Jing; Liu, Lei; Yu, Tao; Cui, Yongqi; Deng, Wei; Yan, Mingxia; Li, Jinjun; Yao, Ming
2014-05-06
To identify cancer-related proteins, we used isobaric tags in a relative and absolute quantitation (iTRAQ) proteomic approach and SWATH™ quantification approach to analyze the secretome of an isogenic pair of highly metastatic and low metastatic non-small-cell lung cancer (NSCLC) cell lines. In addition, we compared two groups of pooled serum samples (12 early-stage and 12 late-stage patients) to mine data for candidates screened by iTRAQ-labeled proteomic analysis. A total of 110 proteins and 71 proteins were observed to be significantly differentially expressed in the cell line secretome and NSCLC sera, respectively. Among these proteins, CD109 was found to be highly expressed in both the highly metastatic cell line secretome and the group of late-stage patients. A sandwich ELISA assay also demonstrated an elevation of serum CD109 levels in individual NSCLC patients (n=30) compared with healthy subjects (n=19). Furthermore, CD109 displayed higher expression in lung cancer tissues compared with their matched noncancerous lung tissues (n=72). In addition, the knockdown of CD109 influenced several NSCLC cell bio-functions, for instance, depressing cell growth, affecting cell cycle phases. These phenomena suggest that CD109 plays a critical role in NSCLC progression. We simultaneously applied two quantitative proteomic approaches-iTRAQ-labeling and SWATH™-to analyze the secretome of metastatic cell lines, in order to explore the cancer-associated proteins in conditioned media. In this study, our results indicate that CD109 plays a critical role in non-small-cell lung cancer (NSCLC) progression, and is overexpressed in advanced NSCLC. Copyright © 2014 Elsevier B.V. All rights reserved.
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Van, Phu T; Schmid, Amy K; King, Nichole L; Kaur, Amardeep; Pan, Min; Whitehead, Kenia; Koide, Tie; Facciotti, Marc T; Goo, Young Ah; Deutsch, Eric W; Reiss, David J; Mallick, Parag; Baliga, Nitin S
2008-09-01
The relatively small numbers of proteins and fewer possible post-translational modifications in microbes provide a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a PeptideAtlas (PA) covering 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636 000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has highlighted plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore, we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics.
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Li, Qingye; Chang, Rong; Sun, Yijun; Li, Bosheng
2016-01-01
Low temperature (LT) is one of the most important abiotic stresses that can significantly reduce crop yield. To gain insight into how Spirulina responds to LT stress, comprehensive physiological and proteomic analyses were conducted in this study. Significant decreases in growth and pigment levels as well as excessive accumulation of compatible osmolytes were observed in response to LT stress. An isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics approach was used to identify changes in protein abundance in Spirulina under LT. A total of 3,782 proteins were identified, of which 1,062 showed differential expression. Bioinformatics analysis indicated that differentially expressed proteins that were enriched in photosynthesis, carbohydrate metabolism, amino acid biosynthesis, and translation are important for the maintenance of cellular homeostasis and metabolic balance in Spirulina when subjected to LT stress. The up-regulation of proteins involved in gluconeogenesis, starch and sucrose metabolism, and amino acid biosynthesis served as coping mechanisms of Spirulina in response to LT stress. Moreover, the down-regulated expression of proteins involved in glycolysis, TCA cycle, pentose phosphate pathway, photosynthesis, and translation were associated with reduced energy consumption. The findings of the present study allow a better understanding of the response of Spirulina to LT stress and may facilitate in the elucidation of mechanisms underlying LT tolerance. PMID:27902743
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Li, Qingye; Chang, Rong; Sun, Yijun; Li, Bosheng
2016-01-01
Low temperature (LT) is one of the most important abiotic stresses that can significantly reduce crop yield. To gain insight into how Spirulina responds to LT stress, comprehensive physiological and proteomic analyses were conducted in this study. Significant decreases in growth and pigment levels as well as excessive accumulation of compatible osmolytes were observed in response to LT stress. An isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics approach was used to identify changes in protein abundance in Spirulina under LT. A total of 3,782 proteins were identified, of which 1,062 showed differential expression. Bioinformatics analysis indicated that differentially expressed proteins that were enriched in photosynthesis, carbohydrate metabolism, amino acid biosynthesis, and translation are important for the maintenance of cellular homeostasis and metabolic balance in Spirulina when subjected to LT stress. The up-regulation of proteins involved in gluconeogenesis, starch and sucrose metabolism, and amino acid biosynthesis served as coping mechanisms of Spirulina in response to LT stress. Moreover, the down-regulated expression of proteins involved in glycolysis, TCA cycle, pentose phosphate pathway, photosynthesis, and translation were associated with reduced energy consumption. The findings of the present study allow a better understanding of the response of Spirulina to LT stress and may facilitate in the elucidation of mechanisms underlying LT tolerance.
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Schilling, Birgit; Rardin, Matthew J; MacLean, Brendan X; Zawadzka, Anna M; Frewen, Barbara E; Cusack, Michael P; Sorensen, Dylan J; Bereman, Michael S; Jing, Enxuan; Wu, Christine C; Verdin, Eric; Kahn, C Ronald; Maccoss, Michael J; Gibson, Bradford W
2012-05-01
Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models.
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Highly Reproducible Label Free Quantitative Proteomic Analysis of RNA Polymerase Complexes*
Mosley, Amber L.; Sardiu, Mihaela E.; Pattenden, Samantha G.; Workman, Jerry L.; Florens, Laurence; Washburn, Michael P.
2011-01-01
The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports. PMID
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The quantitative and condition-dependent Escherichia coli proteome
Schmidt, Alexander; Kochanowski, Karl; Vedelaar, Silke; Ahrné, Erik; Volkmer, Benjamin; Callipo, Luciano; Knoops, Kèvin; Bauer, Manuel; Aebersold, Ruedi; Heinemann, Matthias
2016-01-01
Measuring precise concentrations of proteins can provide insights into biological processes. Here, we use efficient protein extraction and sample fractionation and state-of-the-art quantitative mass spectrometry techniques to generate a comprehensive, condition-dependent protein abundance map of Escherichia coli. We measure cellular protein concentrations for 55% of predicted E. coli genes (>2300 proteins) under 22 different experimental conditions and identify methylation and N-terminal protein acetylations previously not known to be prevalent in bacteria. We uncover system-wide proteome allocation, expression regulation, and post-translational adaptations. These data provide a valuable resource for the systems biology and broader E. coli research communities. PMID:26641532
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Lee, Ji Yoon; Pajarillo, Edward Alain B; Kim, Min Jeong; Chae, Jong Pyo; Kang, Dae-Kyung
2013-01-04
Lactobacillus johnsonii PF01 has been reported to be highly resistant to bile, a key property of probiotic microorganisms. Here, we examine the nature of the bile-salt tolerance of L. johnsonii PF01. Growth inhibition and surface morphology and physiology aberrations were observed after overnight exposure to bile stress. Quantitative proteomic profiles using iTRAQ-LC-MS/MS technology identified 8307 peptides from both untreated PF01 cells and those exposed to 0.1%, 0.2%, and 0.3% bile salts. Some 215 proteins exhibited changed levels in response to bile stress; of these, levels of 94 peptides increased while those of 121 decreased. These were classified into the following categories: stress responses, cell division, transcription, translation, nucleotide metabolism, carbohydrate transport and metabolism, cell wall biosynthesis, and amino acid biosynthesis, and 16 of unidentified function. Analysis of the mRNA expression of selected genes by quantitative reverse transcriptase-PCR verified the proteomic data. Both proteomic and mRNA data provided evidence for increased phosphotransferase activity and cell wall biosynthesis. In addition, three bile salt hydrolases were significantly upregulated by bile exposure. These findings provide a basis for future evaluations of the tolerance of potential probiotic strains toward the various gastrointestinal challenges, including bile stress.
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Unraveling Molecular Differences of Gastric Cancer by Label-Free Quantitative Proteomics Analysis.
Dai, Peng; Wang, Qin; Wang, Weihua; Jing, Ruirui; Wang, Wei; Wang, Fengqin; Azadzoi, Kazem M; Yang, Jing-Hua; Yan, Zhen
2016-01-21
Gastric cancer (GC) has significant morbidity and mortality worldwide and especially in China. Its molecular pathogenesis has not been thoroughly elaborated. The acknowledged biomarkers for diagnosis, prognosis, recurrence monitoring and treatment are lacking. Proteins from matched pairs of human GC and adjacent tissues were analyzed by a coupled label-free Mass Spectrometry (MS) approach, followed by functional annotation with software analysis. Nano-LC-MS/MS, quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry were used to validate dysregulated proteins. One hundred forty-six dysregulated proteins with more than twofold expressions were quantified, 22 of which were first reported to be relevant with GC. Most of them were involved in cancers and gastrointestinal disease. The expression of a panel of four upregulated nucleic acid binding proteins, heterogeneous nuclear ribonucleoprotein hnRNPA2B1, hnRNPD, hnRNPL and Y-box binding protein 1 (YBX-1) were validated by Nano-LC-MS/MS, qRT-PCR, western blot and immunohistochemistry assays in ten GC patients' tissues. They were located in the keynotes of a predicted interaction network and might play important roles in abnormal cell growth. The label-free quantitative proteomic approach provides a deeper understanding and novel insight into GC-related molecular changes and possible mechanisms. It also provides some potential biomarkers for clinical diagnosis.
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Liberato, Tarcísio; Troncone, Lanfranco Ranieri Paolo; Yamashiro, Edson T; Serrano, Solange M T; Zelanis, André
2016-03-01
Here we present a proteomic characterization of Phoneutria nigriventer venom. A shotgun proteomic approach allowed the identification, for the first time, of O-glycosyl hydrolases (chitinases) in P. nigriventer venom. The electrophoretic profiles under nonreducing and reducing conditions, and protein identification by mass spectrometry, indicated the presence of oligomeric toxin structures in the venom. Complementary proteomic approaches allowed for a qualitative and semi-quantitative profiling of P. nigriventer venom complexity, expanding its known venom proteome diversity.
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Zhang, Qiang; Cundiff, Judy K.; Maria, Sarah D.; McMahon, Robert J.; Woo, Jessica G.; Davidson, Barbara S.; Morrow, Ardythe L.
2013-01-01
In-depth understanding of the changing functions of human milk (HM) proteins and the corresponding physiological adaptions of the lactating mammary gland has been inhibited by incomplete knowledge of the HM proteome. We analyzed the HM whey proteome (n = 10 women with samples at 1 week and 1, 3, 6, 9 and 12 months) using a quantitative proteomic approach. One thousand three hundred and thirty three proteins were identified with 615 being quantified. Principal component analysis revealed a transition in the HM whey proteome-throughout the first year of lactation. Abundance changes in IgG, sIgA and sIgM display distinct features during the first year. Complement components and other acute-phase proteins are generally at higher levels in early lactation. Proteomic analysis further suggests that the sources of milk fatty acids (FA) shift from more direct blood influx to more de novo mammary synthesis over lactation. The abundances of the majority of glycoproteins decline over lactation, which is consistent with increased enzyme expression in glycoprotein degradation and decreased enzyme expression in glycoprotein synthesis. Cellular detoxification machinery may be transformed as well, thereby accommodating increased metabolic activities in late lactation. The multiple developing functions of HM proteins and the corresponding mammary adaption become more apparent from this study. PMID:28250401
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Quantitative proteomic analysis of the Salmonella-lettuce interaction
Zhang, Yuping; Nandakumar, Renu; Bartelt-Hunt, Shannon L; Snow, Daniel D; Hodges, Laurie; Li, Xu
2014-01-01
Human pathogens can internalize food crops through root and surface uptake and persist inside crop plants. The goal of the study was to elucidate the global modulation of bacteria and plant protein expression after Salmonella internalizes lettuce. A quantitative proteomic approach was used to analyse the protein expression of Salmonella enterica serovar Infantis and lettuce cultivar Green Salad Bowl 24 h after infiltrating S. Infantis into lettuce leaves. Among the 50 differentially expressed proteins identified by comparing internalized S. Infantis against S. Infantis grown in Luria Broth, proteins involved in glycolysis were down-regulated, while one protein involved in ascorbate uptake was up-regulated. Stress response proteins, especially antioxidant proteins, were up-regulated. The modulation in protein expression suggested that internalized S. Infantis might utilize ascorbate as a carbon source and require multiple stress response proteins to cope with stresses encountered in plants. On the other hand, among the 20 differentially expressed lettuce proteins, proteins involved in defense response to bacteria were up-regulated. Moreover, the secreted effector PipB2 of S. Infantis and R proteins of lettuce were induced after bacterial internalization into lettuce leaves, indicating human pathogen S. Infantis triggered the defense mechanisms of lettuce, which normally responds to plant pathogens. PMID:24512637
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Megger, Dominik Andre; Rosowski, Kristin; Ahrens, Maike; Bracht, Thilo; Eisenacher, Martin; Schlaak, Jörg F; Weber, Frank; Hoffmann, Andreas-Claudius; Meyer, Helmut E; Baba, Hideo A; Sitek, Barbara
2017-03-01
Human hepatocellular carcinoma (HCC) is a severe malignant disease, and accurate and reliable diagnostic markers are still needed. This study was aimed for the discovery of novel marker candidates by quantitative proteomics. Proteomic differences between HCC and nontumorous liver tissue were studied by mass spectrometry. Among several significantly upregulated proteins, translocator protein 18 (TSPO) and Ras-related protein Rab-1A (RAB1A) were selected for verification by immunohistochemistry in an independent cohort. For RAB1A, a high accuracy for the discrimination of HCC and nontumorous liver tissue was observed. RAB1A was verified to be a potent biomarker candidate for HCC.
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The speciation of the proteome
Jungblut, Peter R; Holzhütter, Hermann G; Apweiler, Rolf; Schlüter, Hartmut
2008-01-01
Introduction In proteomics a paradox situation developed in the last years. At one side it is basic knowledge that proteins are post-translationally modified and occur in different isoforms. At the other side the protein expression concept disclaims post-translational modifications by connecting protein names directly with function. Discussion Optimal proteome coverage is today reached by bottom-up liquid chromatography/mass spectrometry. But quantification at the peptide level in shotgun or bottom-up approaches by liquid chromatography and mass spectrometry is completely ignoring that a special peptide may exist in an unmodified form and in several-fold modified forms. The acceptance of the protein species concept is a basic prerequisite for meaningful quantitative analyses in functional proteomics. In discovery approaches only top-down analyses, separating the protein species before digestion, identification and quantification by two-dimensional gel electrophoresis or protein liquid chromatography, allow the correlation between changes of a biological situation and function. Conclusion To obtain biological relevant information kinetics and systems biology have to be performed at the protein species level, which is the major challenge in proteomics today. PMID:18638390
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Wei, Zhe; Hu, Wei; Lin, Qishan; Cheng, Xiaoyan; Tong, Mengjie; Zhu, Lili; Chen, Rongzhi; He, Guangcun
2009-05-01
Engineering and breeding resistant plant varieties are the most effective and environmentally friendly ways to control agricultural pests and improve crop performance. However, the mechanism of plant resistance to pests is poorly understood. Here we used a quantitative mass-spectrometry-based proteomic approach for comparative analysis of expression profiles of proteins in rice leaf sheaths in responses to infestation by the brown planthopper (Nilaparvata lugens Stål, BPH), which is a serious rice crop pest. Proteins involved in multiple pathways showed significant changes in expression in response to BPH feeding, including jasmonic acid synthesis proteins, oxidative stress response proteins, beta-glucanases, protein; kinases, clathrin protein, glycine cleavage system protein, photosynthesis proteins and aquaporins. The corresponding genes of eight important proteins were further analyzed by quantitative RT-PCR. Proteomic and transcript responses that were related to wounding, oxidative and pathogen stress overlapped considerably between BPH-resistant (carrying the resistance gene BPH15) and susceptible rice lines. In contrast, proteins and genes related to callose metabolism remained unchanged and glycine cleavage system protein was up-regulated in the BPH-resistant lines, indicating that they have an efficient and specific defense mechanism. Our results provide new information about the interaction between rice and the BPH.
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Click-MS: Tagless Protein Enrichment Using Bioorthogonal Chemistry for Quantitative Proteomics.
Smits, Arne H; Borrmann, Annika; Roosjen, Mark; van Hest, Jan C M; Vermeulen, Michiel
2016-12-16
Epitope-tagging is an effective tool to facilitate protein enrichment from crude cell extracts. Traditionally, N- or C-terminal fused tags are employed, which, however, can perturb protein function. Unnatural amino acids (UAAs) harboring small reactive handles can be site-specifically incorporated into proteins, thus serving as a potential alternative for conventional protein tags. Here, we introduce Click-MS, which combines the power of site-specific UAA incorporation, bioorthogonal chemistry, and quantitative mass spectrometry-based proteomics to specifically enrich a single protein of interest from crude mammalian cell extracts. By genetic encoding of p-azido-l-phenylalanine, the protein of interest can be selectively captured using copper-free click chemistry. We use Click-MS to enrich proteins that function in different cellular compartments, and we identify protein-protein interactions, showing the great potential of Click-MS for interaction proteomics workflows.
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Choi, Hyungwon; Kim, Sinae; Fermin, Damian; Tsou, Chih-Chiang; Nesvizhskii, Alexey I
2015-11-03
We introduce QPROT, a statistical framework and computational tool for differential protein expression analysis using protein intensity data. QPROT is an extension of the QSPEC suite, originally developed for spectral count data, adapted for the analysis using continuously measured protein-level intensity data. QPROT offers a new intensity normalization procedure and model-based differential expression analysis, both of which account for missing data. Determination of differential expression of each protein is based on the standardized Z-statistic based on the posterior distribution of the log fold change parameter, guided by the false discovery rate estimated by a well-known Empirical Bayes method. We evaluated the classification performance of QPROT using the quantification calibration data from the clinical proteomic technology assessment for cancer (CPTAC) study and a recently published Escherichia coli benchmark dataset, with evaluation of FDR accuracy in the latter. QPROT is a statistical framework with computational software tool for comparative quantitative proteomics analysis. It features various extensions of QSPEC method originally built for spectral count data analysis, including probabilistic treatment of missing values in protein intensity data. With the increasing popularity of label-free quantitative proteomics data, the proposed method and accompanying software suite will be immediately useful for many proteomics laboratories. This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.
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Yu, Kebing; Salomon, Arthur R
2009-12-01
Recently, dramatic progress has been achieved in expanding the sensitivity, resolution, mass accuracy, and scan rate of mass spectrometers able to fragment and identify peptides through MS/MS. Unfortunately, this enhanced ability to acquire proteomic data has not been accompanied by a concomitant increase in the availability of flexible tools allowing users to rapidly assimilate, explore, and analyze this data and adapt to various experimental workflows with minimal user intervention. Here we fill this critical gap by providing a flexible relational database called PeptideDepot for organization of expansive proteomic data sets, collation of proteomic data with available protein information resources, and visual comparison of multiple quantitative proteomic experiments. Our software design, built upon the synergistic combination of a MySQL database for safe warehousing of proteomic data with a FileMaker-driven graphical user interface for flexible adaptation to diverse workflows, enables proteomic end-users to directly tailor the presentation of proteomic data to the unique analysis requirements of the individual proteomics lab. PeptideDepot may be deployed as an independent software tool or integrated directly with our high throughput autonomous proteomic pipeline used in the automated acquisition and post-acquisition analysis of proteomic data.
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Terfve, Camille; Sabidó, Eduard; Wu, Yibo; Gonçalves, Emanuel; Choi, Meena; Vaga, Stefania; Vitek, Olga; Saez-Rodriguez, Julio; Aebersold, Ruedi
2017-02-03
Advances in mass spectrometry have made the quantitative measurement of proteins across multiple samples a reality, allowing for the study of complex biological systems such as the metabolic syndrome. Although the deregulation of lipid metabolism and increased hepatic storage of triacylglycerides are known to play a part in the onset of the metabolic syndrome, its molecular basis and dependency on dietary and genotypic factors are poorly characterized. Here, we used an experimental design with two different mouse strains and dietary and metabolic perturbations to generate a compendium of quantitative proteome data using three mass spectrometric techniques. The data reproduce known properties of the metabolic system and indicate differential molecular adaptation of the two mouse strains to perturbations, contributing to a better understanding of the metabolic syndrome. We show that high-quality, high-throughput proteomic data sets provide an unbiased broad overview of the behavior of complex systems after perturbation.
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The role of proteomics in studies of protein moonlighting.
Beynon, Robert J; Hammond, Dean; Harman, Victoria; Woolerton, Yvonne
2014-12-01
The increasing acceptance that proteins may exert multiple functions in the cell brings with it new analytical challenges that will have an impact on the field of proteomics. Many proteomics workflows begin by destroying information about the interactions between different proteins, and the reduction of a complex protein mixture to constituent peptides also scrambles information about the combinatorial potential of post-translational modifications. To bring the focus of proteomics on to the domain of protein moonlighting will require novel analytical and quantitative approaches.
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Teaching Expression Proteomics: From the Wet-Lab to the Laptop
ERIC Educational Resources Information Center
Teixeira, Miguel C.; Santos, Pedro M.; Rodrigues, Catarina; Sa-Correia, Isabel
2009-01-01
Expression proteomics has become, in recent years, a key genome-wide expression approach in fundamental and applied life sciences. This postgenomic technology aims the quantitative analysis of all the proteins or protein forms (the so-called proteome) of a given organism in a given environmental and genetic context. It is a challenge to provide…
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Gadher, Suresh Jivan; Marczak, Łukasz; Łuczak, Magdalena; Stobiecki, Maciej; Widlak, Piotr; Kovarova, Hana
2016-01-01
Every year since 2007, the Central and Eastern European Proteomic Conference (CEEPC) has excelled in representing state-of-the-art proteomics in and around Central and Eastern Europe, and linking it to international institutions worldwide. Its mission remains to contribute to all approaches of proteomics including traditional and often-revisited methodologies as well as the latest technological achievements in clinical, quantitative and structural proteomics with a view to systems biology of a variety of processes. The 9th CEEPC was held from June 15th to 18th, 2015, at the Institute of Bioorganic Chemistry, Polish Academy of Sciences in Poznań, Poland. The scientific program stimulated exchange of proteomic knowledge whilst the spectacular venue of the conference allowed participants to enjoy the cobblestoned historical city of Poznań.
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Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan
2016-01-29
Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics.
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A proteomic approach to obesity and type 2 diabetes
López-Villar, Elena; Martos-Moreno, Gabriel Á; Chowen, Julie A; Okada, Shigeru; Kopchick, John J; Argente, Jesús
2015-01-01
The incidence of obesity and type diabetes 2 has increased dramatically resulting in an increased interest in its biomedical relevance. However, the mechanisms that trigger the development of diabetes type 2 in obese patients remain largely unknown. Scientific, clinical and pharmaceutical communities are dedicating vast resources to unravel this issue by applying different omics tools. During the last decade, the advances in proteomic approaches and the Human Proteome Organization have opened and are opening a new door that may be helpful in the identification of patients at risk and to improve current therapies. Here, we briefly review some of the advances in our understanding of type 2 diabetes that have occurred through the application of proteomics. We also review, in detail, the current improvements in proteomic methodologies and new strategies that could be employed to further advance our understanding of this pathology. By applying these new proteomic advances, novel therapeutic and/or diagnostic protein targets will be discovered in the obesity/Type 2 diabetes area. PMID:25960181
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ABRF-PRG07: advanced quantitative proteomics study.
Falick, Arnold M; Lane, William S; Lilley, Kathryn S; MacCoss, Michael J; Phinney, Brett S; Sherman, Nicholas E; Weintraub, Susan T; Witkowska, H Ewa; Yates, Nathan A
2011-04-01
A major challenge for core facilities is determining quantitative protein differences across complex biological samples. Although there are numerous techniques in the literature for relative and absolute protein quantification, the majority is nonroutine and can be challenging to carry out effectively. There are few studies comparing these technologies in terms of their reproducibility, accuracy, and precision, and no studies to date deal with performance across multiple laboratories with varied levels of expertise. Here, we describe an Association of Biomolecular Resource Facilities (ABRF) Proteomics Research Group (PRG) study based on samples composed of a complex protein mixture into which 12 known proteins were added at varying but defined ratios. All of the proteins were present at the same concentration in each of three tubes that were provided. The primary goal of this study was to allow each laboratory to evaluate its capabilities and approaches with regard to: detection and identification of proteins spiked into samples that also contain complex mixtures of background proteins and determination of relative quantities of the spiked proteins. The results returned by 43 participants were compiled by the PRG, which also collected information about the strategies used to assess overall performance and as an aid to development of optimized protocols for the methodologies used. The most accurate results were generally reported by the most experienced laboratories. Among laboratories that used the same technique, values that were closer to the expected ratio were obtained by more experienced groups.
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Quantitative Proteomics of the Infectious and Replicative Forms of Chlamydia trachomatis
Skipp, Paul J. S.; Hughes, Chris; McKenna, Thérèse; Edwards, Richard; Langridge, James; Thomson, Nicholas R.; Clarke, Ian N.
2016-01-01
The obligate intracellular developmental cycle of Chlamydia trachomatis presents significant challenges in defining its proteome. In this study we have applied quantitative proteomics to both the intracellular reticulate body (RB) and the extracellular elementary body (EB) from C. trachomatis. We used C. trachomatis L2 as a model chlamydial isolate for our study since it has a high infectivity:particle ratio and there is an excellent quality genome sequence. EBs and RBs (>99% pure) were quantified by chromosomal and plasmid copy number using PCR, from which the concentrations of chlamydial proteins per bacterial cell/genome were determined. RBs harvested at 15h post infection (PI) were purified by three successive rounds of gradient centrifugation. This is the earliest possible time to obtain purified RBs, free from host cell components in quantity, within the constraints of the technology. EBs were purified at 48h PI. We then used two-dimensional reverse phase UPLC to fractionate RB or EB peptides before mass spectroscopic analysis, providing absolute amount estimates of chlamydial proteins. The ability to express the data as molecules per cell gave ranking in both abundance and energy requirements for synthesis, allowing meaningful identification of rate-limiting components. The study assigned 562 proteins with high confidence and provided absolute estimates of protein concentration for 489 proteins. Interestingly, the data showed an increase in TTS capacity at 15h PI. Most of the enzymes involved in peptidoglycan biosynthesis were detected along with high levels of muramidase (in EBs) suggesting breakdown of peptidoglycan occurs in the non-dividing form of the microorganism. All the genome-encoded enzymes for glycolysis, pentose phosphate pathway and tricarboxylic acid cycle were identified and quantified; these data supported the observation that the EB is metabolically active. The availability of detailed, accurate quantitative proteomic data will be
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Quantitative Proteomics by Metabolic Labeling of Model Organisms*
Gouw, Joost W.; Krijgsveld, Jeroen; Heck, Albert J. R.
2010-01-01
In the biological sciences, model organisms have been used for many decades and have enabled the gathering of a large proportion of our present day knowledge of basic biological processes and their derailments in disease. Although in many of these studies using model organisms, the focus has primarily been on genetics and genomics approaches, it is important that methods become available to extend this to the relevant protein level. Mass spectrometry-based proteomics is increasingly becoming the standard to comprehensively analyze proteomes. An important transition has been made recently by moving from charting static proteomes to monitoring their dynamics by simultaneously quantifying multiple proteins obtained from differently treated samples. Especially the labeling with stable isotopes has proved an effective means to accurately determine differential expression levels of proteins. Among these, metabolic incorporation of stable isotopes in vivo in whole organisms is one of the favored strategies. In this perspective, we will focus on methodologies to stable isotope label a variety of model organisms in vivo, ranging from relatively simple organisms such as bacteria and yeast to Caenorhabditis elegans, Drosophila, and Arabidopsis up to mammals such as rats and mice. We also summarize how this has opened up ways to investigate biological processes at the protein level in health and disease, revealing conservation and variation across the evolutionary tree of life. PMID:19955089
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Computational approaches to protein inference in shotgun proteomics
2012-01-01
Shotgun proteomics has recently emerged as a powerful approach to characterizing proteomes in biological samples. Its overall objective is to identify the form and quantity of each protein in a high-throughput manner by coupling liquid chromatography with tandem mass spectrometry. As a consequence of its high throughput nature, shotgun proteomics faces challenges with respect to the analysis and interpretation of experimental data. Among such challenges, the identification of proteins present in a sample has been recognized as an important computational task. This task generally consists of (1) assigning experimental tandem mass spectra to peptides derived from a protein database, and (2) mapping assigned peptides to proteins and quantifying the confidence of identified proteins. Protein identification is fundamentally a statistical inference problem with a number of methods proposed to address its challenges. In this review we categorize current approaches into rule-based, combinatorial optimization and probabilistic inference techniques, and present them using integer programing and Bayesian inference frameworks. We also discuss the main challenges of protein identification and propose potential solutions with the goal of spurring innovative research in this area. PMID:23176300
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Proteomic approaches and their application to plant gravitropism.
Basu, Proma; Luesse, Darron R; Wyatt, Sarah E
2015-01-01
Proteomics is a powerful technique that allows researchers a window into how an organism responds to a mutation, a specific environment, or at a distinct point during development by quantifying relative protein abundance and posttranslational modifications. Here, we describe methods for the proteomic analysis of Arabidopsis thaliana tissue. Extraction protocols are provided for isolation of soluble, plasma membrane, and tonoplast proteins. In addition, basic analysis and quality metrics for MS/MS data are discussed. The protocols outlined have the potential to unlock new avenues of research that are not possible through basic genetics or transcriptomic approaches. By combining proteomic information with known gene regulatory patterns, researchers can gain a complete picture of how molecular pathways, such as those required for gravitropism, are initiated, regulated, and terminated.
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Quantitative Proteomics Analysis of Inborn Errors of Cholesterol Synthesis
Jiang, Xiao-Sheng; Backlund, Peter S.; Wassif, Christopher A.; Yergey, Alfred L.; Porter, Forbes D.
2010-01-01
Smith-Lemli-Opitz syndrome (SLOS) and lathosterolosis are malformation syndromes with cognitive deficits caused by mutations of 7-dehydrocholesterol reductase (DHCR7) and lathosterol 5-desaturase (SC5D), respectively. DHCR7 encodes the last enzyme in the Kandutsch-Russel cholesterol biosynthetic pathway, and impaired DHCR7 activity leads to a deficiency of cholesterol and an accumulation of 7-dehydrocholesterol. SC5D catalyzes the synthesis of 7-dehydrocholesterol from lathosterol. Impaired SC5D activity leads to a similar deficiency of cholesterol but an accumulation of lathosterol. Although the genetic and biochemical causes underlying both syndromes are known, the pathophysiological processes leading to the developmental defects remain unclear. To study the pathophysiological mechanisms underlying SLOS and lathosterolosis neurological symptoms, we performed quantitative proteomics analysis of SLOS and lathosterolosis mouse brain tissue and identified multiple biological pathways affected in Dhcr7Δ3–5/Δ3–5 and Sc5d−/− E18.5 embryos. These include alterations in mevalonate metabolism, apoptosis, glycolysis, oxidative stress, protein biosynthesis, intracellular trafficking, and cytoskeleton. Comparison of proteome alterations in both Dhcr7Δ3–5/Δ3–5 and Sc5d−/− brain tissues helps elucidate whether perturbed protein expression was due to decreased cholesterol or a toxic effect of sterol precursors. Validation of the proteomics results confirmed increased expression of isoprenoid and cholesterol synthetic enzymes. This alteration of isoprenoid synthesis may underlie the altered posttranslational modification of Rab7, a small GTPase that is functionally dependent on prenylation with geranylgeranyl, that we identified and validated in this study. These data suggested that although cholesterol synthesis is impaired in both Dhcr7Δ3–5/Δ3–5 and Sc5d−/− embryonic brain tissues the synthesis of nonsterol isoprenoids may be increased and thus
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Quantitative proteomics of bronchoalveolar lavage fluid in lung adenocarcinoma.
Almatroodi, Saleh A; McDonald, Christine F; Collins, Allison L; Darby, Ian A; Pouniotis, Dodie S
2015-01-01
The most commonly reported primary lung cancer subtype is adenocarcinoma, which is associated with a poor prognosis and short survival. Proteomic studies on human body fluids such as bronchoalveolar lavage fluid (BALF) have become essential methods for biomarker discovery, examination of tumor pathways and investigation of potential treatments. This study used quantitative proteomics to investigate the up-regulation of novel proteins in BALF from patients with primary lung adenocarcinoma in order to identify potential biomarkers. BALF samples from individuals with and without primary lung adenocarcinoma were analyzed using liquid chromatography-mass spectrometry. One thousand and one hundred proteins were identified, 33 of which were found to be consistently overexpressed in all lung adenocarcinoma samples compared to non-cancer controls. A number of overexpressed proteins have been previously shown to be related to lung cancer progression including S100-A8, annexin A1, annexin A2, thymidine phosphorylase and transglutaminase 2. The overexpression of a number of specific proteins in BALF from patients with primary lung adenocarcinoma may be used as a potential biomarker for lung adenocarcinoma. Copyright© 2015, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.
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Pan, Sheng; Rush, John; Peskind, Elaine R; Galasko, Douglas; Chung, Kathryn; Quinn, Joseph; Jankovic, Joseph; Leverenz, James B; Zabetian, Cyrus; Pan, Catherine; Wang, Yan; Oh, Jung Hun; Gao, Jean; Zhang, Jianpeng; Montine, Thomas; Zhang, Jing
2008-02-01
Targeted quantitative proteomics by mass spectrometry aims to selectively detect one or a panel of peptides/proteins in a complex sample and is particularly appealing for novel biomarker verification/validation because it does not require specific antibodies. Here, we demonstrated the application of targeted quantitative proteomics in searching, identifying, and quantifying selected peptides in human cerebrospinal spinal fluid (CSF) using a matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (MALDI TOF/TOF)-based platform. The approach involved two major components: the use of isotopic-labeled synthetic peptides as references for targeted identification and quantification and a highly selective mass spectrometric analysis based on the unique characteristics of the MALDI instrument. The platform provides high confidence for targeted peptide detection in a complex system and can potentially be developed into a high-throughput system. Using the liquid chromatography (LC) MALDI TOF/TOF platform and the complementary identification strategy, we were able to selectively identify and quantify a panel of targeted peptides in the whole proteome of CSF without prior depletion of abundant proteins. The effectiveness and robustness of the approach associated with different sample complexity, sample preparation strategies, as well as mass spectrometric quantification were evaluated. Other issues related to chromatography separation and the feasibility for high-throughput analysis were also discussed. Finally, we applied targeted quantitative proteomics to analyze a subset of previously identified candidate markers in CSF samples of patients with Parkinson's disease (PD) at different stages and Alzheimer's disease (AD) along with normal controls.
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Li, Tie-Mei; Zhang, Ju-en; Lin, Rui; Chen, She; Luo, Minmin; Dong, Meng-Qiu
2016-01-01
Sleep is a ubiquitous, tightly regulated, and evolutionarily conserved behavior observed in almost all animals. Prolonged sleep deprivation can be fatal, indicating that sleep is a physiological necessity. However, little is known about its core function. To gain insight into this mystery, we used advanced quantitative proteomics technology to survey the global changes in brain protein abundance. Aiming to gain a comprehensive profile, our proteomics workflow included filter-aided sample preparation (FASP), which increased the coverage of membrane proteins; tandem mass tag (TMT) labeling, for relative quantitation; and high resolution, high mass accuracy, high throughput mass spectrometry (MS). In total, we obtained the relative abundance ratios of 9888 proteins encoded by 6070 genes. Interestingly, we observed significant enrichment for mitochondrial proteins among the differentially expressed proteins. This finding suggests that sleep deprivation strongly affects signaling pathways that govern either energy metabolism or responses to mitochondrial stress. Additionally, the differentially-expressed proteins are enriched in pathways implicated in age-dependent neurodegenerative diseases, including Parkinson’s, Huntington’s, and Alzheimer’s, hinting at possible connections between sleep loss, mitochondrial stress, and neurodegeneration. PMID:27684481
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Quantitative Proteomics Identifies Activation of Hallmark Pathways of Cancer in Patient Melanoma.
Byrum, Stephanie D; Larson, Signe K; Avaritt, Nathan L; Moreland, Linley E; Mackintosh, Samuel G; Cheung, Wang L; Tackett, Alan J
2013-03-01
Molecular pathways regulating melanoma initiation and progression are potential targets of therapeutic development for this aggressive cancer. Identification and molecular analysis of these pathways in patients has been primarily restricted to targeted studies on individual proteins. Here, we report the most comprehensive analysis of formalin-fixed paraffin-embedded human melanoma tissues using quantitative proteomics. From 61 patient samples, we identified 171 proteins varying in abundance among benign nevi, primary melanoma, and metastatic melanoma. Seventy-three percent of these proteins were validated by immunohistochemistry staining of malignant melanoma tissues from the Human Protein Atlas database. Our results reveal that molecular pathways involved with tumor cell proliferation, motility, and apoptosis are mis-regulated in melanoma. These data provide the most comprehensive proteome resource on patient melanoma and reveal insight into the molecular mechanisms driving melanoma progression.
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Yu, Kebing; Salomon, Arthur R.
2010-01-01
Recently, dramatic progress has been achieved in expanding the sensitivity, resolution, mass accuracy, and scan rate of mass spectrometers able to fragment and identify peptides through tandem mass spectrometry (MS/MS). Unfortunately, this enhanced ability to acquire proteomic data has not been accompanied by a concomitant increase in the availability of flexible tools allowing users to rapidly assimilate, explore, and analyze this data and adapt to a variety of experimental workflows with minimal user intervention. Here we fill this critical gap by providing a flexible relational database called PeptideDepot for organization of expansive proteomic data sets, collation of proteomic data with available protein information resources, and visual comparison of multiple quantitative proteomic experiments. Our software design, built upon the synergistic combination of a MySQL database for safe warehousing of proteomic data with a FileMaker-driven graphical user interface for flexible adaptation to diverse workflows, enables proteomic end-users to directly tailor the presentation of proteomic data to the unique analysis requirements of the individual proteomics lab. PeptideDepot may be deployed as an independent software tool or integrated directly with our High Throughput Autonomous Proteomic Pipeline (HTAPP) used in the automated acquisition and post-acquisition analysis of proteomic data. PMID:19834895
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Quantitative Proteomics Analysis of the cAMP/Protein Kinase A Signaling Pathway
2012-01-01
To define the proteins whose expression is regulated by cAMP and protein kinase A (PKA), we used a quantitative proteomics approach in studies of wild-type (WT) and kin- (PKA-null) S49 murine T lymphoma cells. We also compared the impact of endogenous increases in the level of cAMP [by forskolin (Fsk) and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX)] or by a cAMP analogue (8-CPT-cAMP). We identified 1056 proteins in WT and kin- S49 cells and found that 8-CPT-cAMP and Fsk with IBMX produced differences in protein expression. WT S49 cells had a correlation coefficient of 0.41 between DNA microarray data and the proteomics analysis in cells incubated with 8-CPT-cAMP for 24 h and a correlation coefficient of 0.42 between the DNA microarray data obtained at 6 h and the changes in protein expression after incubation with 8-CPT-cAMP for 24 h. Glutathione reductase (Gsr) had a higher level of basal expression in kin- S49 cells than in WT cells. Consistent with this finding, kin- cells are less sensitive to cell killing and generation of malondialdehyde than are WT cells incubated with H2O2. Cyclic AMP acting via PKA thus has a broad impact on protein expression in mammalian cells, including in the regulation of Gsr and oxidative stress. PMID:23110364
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Quantitative Proteomic Profiling of Low-Dose Ionizing Radiation Effects in a Human Skin Model
Hengel, Shawna M.; Aldrich, Joshua T.; Waters, Katrina M.; Pasa-Tolic, Ljiljana; Stenoien, David L.
2014-01-01
To assess responses to low-dose ionizing radiation (LD-IR) exposures potentially encountered during medical diagnostic procedures, nuclear accidents or terrorist acts, a quantitative proteomic approach was used to identify changes in protein abundance in a reconstituted human skin tissue model treated with 0.1 Gy of ionizing radiation. To improve the dynamic range of the assay, subcellular fractionation was employed to remove highly abundant structural proteins and to provide insight into radiation-induced alterations in protein localization. Relative peptide quantification across cellular fractions, control and irradiated samples was performing using 8-plex iTRAQ labeling followed by online two-dimensional nano-scale liquid chromatography and high resolution MS/MS analysis. A total of 107 proteins were detected with statistically significant radiation-induced change in abundance (>1.5 fold) and/or subcellular localization compared to controls. The top biological pathways identified using bioinformatics include organ development, anatomical structure formation and the regulation of actin cytoskeleton. From the proteomic data, a change in proteolytic processing and subcellular localization of the skin barrier protein, filaggrin, was identified, and the results were confirmed by western blotting. This data indicate post-transcriptional regulation of protein abundance, localization and proteolytic processing playing an important role in regulating radiation response in human tissues. PMID:28250387
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Zhang, Cheng-Cheng; Li, Ru; Jiang, Honghui; Lin, Shujun; Rogalski, Jason C; Liu, Kate; Kast, Juergen
2015-02-06
Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.
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Bruderer, Roland; Bernhardt, Oliver M.; Gandhi, Tejas; Miladinović, Saša M.; Cheng, Lin-Yang; Messner, Simon; Ehrenberger, Tobias; Zanotelli, Vito; Butscheid, Yulia; Escher, Claudia; Vitek, Olga; Rinner, Oliver; Reiter, Lukas
2015-01-01
The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP)1-treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD). Our findings imply that DIA should be the preferred method for quantitative protein profiling. PMID:25724911
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Ramus, Claire; Hovasse, Agnès; Marcellin, Marlène; Hesse, Anne-Marie; Mouton-Barbosa, Emmanuelle; Bouyssié, David; Vaca, Sebastian; Carapito, Christine; Chaoui, Karima; Bruley, Christophe; Garin, Jérôme; Cianférani, Sarah; Ferro, Myriam; Van Dorssaeler, Alain; Burlet-Schiltz, Odile; Schaeffer, Christine; Couté, Yohann; Gonzalez de Peredo, Anne
2016-01-30
Proteomic workflows based on nanoLC-MS/MS data-dependent-acquisition analysis have progressed tremendously in recent years. High-resolution and fast sequencing instruments have enabled the use of label-free quantitative methods, based either on spectral counting or on MS signal analysis, which appear as an attractive way to analyze differential protein expression in complex biological samples. However, the computational processing of the data for label-free quantification still remains a challenge. Here, we used a proteomic standard composed of an equimolar mixture of 48 human proteins (Sigma UPS1) spiked at different concentrations into a background of yeast cell lysate to benchmark several label-free quantitative workflows, involving different software packages developed in recent years. This experimental design allowed to finely assess their performances in terms of sensitivity and false discovery rate, by measuring the number of true and false-positive (respectively UPS1 or yeast background proteins found as differential). The spiked standard dataset has been deposited to the ProteomeXchange repository with the identifier PXD001819 and can be used to benchmark other label-free workflows, adjust software parameter settings, improve algorithms for extraction of the quantitative metrics from raw MS data, or evaluate downstream statistical methods. Bioinformatic pipelines for label-free quantitative analysis must be objectively evaluated in their ability to detect variant proteins with good sensitivity and low false discovery rate in large-scale proteomic studies. This can be done through the use of complex spiked samples, for which the "ground truth" of variant proteins is known, allowing a statistical evaluation of the performances of the data processing workflow. We provide here such a controlled standard dataset and used it to evaluate the performances of several label-free bioinformatics tools (including MaxQuant, Skyline, MFPaQ, IRMa-hEIDI and Scaffold) in
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Toward improved peptide feature detection in quantitative proteomics using stable isotope labeling.
Nilse, Lars; Sigloch, Florian Christoph; Biniossek, Martin L; Schilling, Oliver
2015-08-01
Reliable detection of peptides in LC-MS data is a key algorithmic step in the analysis of quantitative proteomics experiments. While highly abundant peptides can be detected reliably by most modern software tools, there is much less agreement on medium and low-intensity peptides in a sample. The choice of software tools can have a big impact on the quantification of proteins, especially for proteins that appear in lower concentrations. However, in many experiments, it is precisely this region of less abundant but substantially regulated proteins that holds the biggest potential for discoveries. This is particularly true for discovery proteomics in the pharmacological sector with a specific interest in key regulatory proteins. In this viewpoint article, we discuss how the development of novel software algorithms allows us to study this region of the proteome with increased confidence. Reliable results are one of many aspects to be considered when deciding on a bioinformatics software platform. Deployment into existing IT infrastructures, compatibility with other software packages, scalability, automation, flexibility, and support need to be considered and are briefly addressed in this viewpoint article. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Combining genomic and proteomic approaches for epigenetics research
Han, Yumiao; Garcia, Benjamin A
2014-01-01
Epigenetics is the study of changes in gene expression or cellular phenotype that do not change the DNA sequence. In this review, current methods, both genomic and proteomic, associated with epigenetics research are discussed. Among them, chromatin immunoprecipitation (ChIP) followed by sequencing and other ChIP-based techniques are powerful techniques for genome-wide profiling of DNA-binding proteins, histone post-translational modifications or nucleosome positions. However, mass spectrometry-based proteomics is increasingly being used in functional biological studies and has proved to be an indispensable tool to characterize histone modifications, as well as DNA–protein and protein–protein interactions. With the development of genomic and proteomic approaches, combination of ChIP and mass spectrometry has the potential to expand our knowledge of epigenetics research to a higher level. PMID:23895656
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Quantitative proteomics reveals the central changes of wheat in response to powdery mildew.
Fu, Ying; Zhang, Hong; Mandal, Siddikun Nabi; Wang, Changyou; Chen, Chunhuan; Ji, Wanquan
2016-01-01
Powdery mildew (Pm), caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most important crop diseases, causing severe economic losses to wheat production worldwide. However, there are few reports about the proteomic response to Bgt infection in resistant wheat. Hence, quantitative proteomic analysis of N9134, a resistant wheat line, was performed to explore the molecular mechanism of wheat in defense against Bgt. Comparing the leaf proteins of Bgt-inoculated N9134 with that of mock-inoculated controls, a total of 2182 protein-species were quantified by iTRAQ at 24, 48 and 72h postinoculation (hpi) with Bgt, of which 394 showed differential accumulation. These differentially accumulated protein-species (DAPs) mainly included pathogenesis-related (PR) polypeptides, oxidative stress responsive proteins and components involved in primary metabolic pathways. KEGG enrichment analysis showed that phenylpropanoid biosynthesis, phenylalanine metabolism and photosynthesis-antenna proteins were the key pathways in response to Bgt infection. InterProScan 5 and the Gibbs Motif Sampler cluster 394 DAPs into eight conserved motifs, which shared leucine repeats and histidine sites in the sequence motifs. Moreover, eight separate protein-protein interaction (PPI) networks were predicted from STRING database. This study provides a powerful platform for further exploration of the molecular mechanism underlying resistant wheat responding to Bgt. Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive pathogenic disease in wheat-producing regions worldwide, resulting in severe yield reductions. Although many resistant wheat varieties have been cultivated, there are few reports about the proteomic response to Bgt infection in resistant wheat. Therefore, an iTRAQ-based quantitative proteomic analysis of a resistant wheat line (N9134) in response to Bgt infection has been performed. This paper provides new insights into the underlying molecular
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EBprot: Statistical analysis of labeling-based quantitative proteomics data.
Koh, Hiromi W L; Swa, Hannah L F; Fermin, Damian; Ler, Siok Ghee; Gunaratne, Jayantha; Choi, Hyungwon
2015-08-01
Labeling-based proteomics is a powerful method for detection of differentially expressed proteins (DEPs). The current data analysis platform typically relies on protein-level ratios, which is obtained by summarizing peptide-level ratios for each protein. In shotgun proteomics, however, some proteins are quantified with more peptides than others, and this reproducibility information is not incorporated into the differential expression (DE) analysis. Here, we propose a novel probabilistic framework EBprot that directly models the peptide-protein hierarchy and rewards the proteins with reproducible evidence of DE over multiple peptides. To evaluate its performance with known DE states, we conducted a simulation study to show that the peptide-level analysis of EBprot provides better receiver-operating characteristic and more accurate estimation of the false discovery rates than the methods based on protein-level ratios. We also demonstrate superior classification performance of peptide-level EBprot analysis in a spike-in dataset. To illustrate the wide applicability of EBprot in different experimental designs, we applied EBprot to a dataset for lung cancer subtype analysis with biological replicates and another dataset for time course phosphoproteome analysis of EGF-stimulated HeLa cells with multiplexed labeling. Through these examples, we show that the peptide-level analysis of EBprot is a robust alternative to the existing statistical methods for the DE analysis of labeling-based quantitative datasets. The software suite is freely available on the Sourceforge website http://ebprot.sourceforge.net/. All MS data have been deposited in the ProteomeXchange with identifier PXD001426 (http://proteomecentral.proteomexchange.org/dataset/PXD001426/). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Quantitative proteomic analysis reveals a simple strategy of global resource allocation in bacteria
Hui, Sheng; Silverman, Josh M; Chen, Stephen S; Erickson, David W; Basan, Markus; Wang, Jilong; Hwa, Terence; Williamson, James R
2015-01-01
A central aim of cell biology was to understand the strategy of gene expression in response to the environment. Here, we study gene expression response to metabolic challenges in exponentially growing Escherichia coli using mass spectrometry. Despite enormous complexity in the details of the underlying regulatory network, we find that the proteome partitions into several coarse-grained sectors, with each sector's total mass abundance exhibiting positive or negative linear relations with the growth rate. The growth rate-dependent components of the proteome fractions comprise about half of the proteome by mass, and their mutual dependencies can be characterized by a simple flux model involving only two effective parameters. The success and apparent generality of this model arises from tight coordination between proteome partition and metabolism, suggesting a principle for resource allocation in proteome economy of the cell. This strategy of global gene regulation should serve as a basis for future studies on gene expression and constructing synthetic biological circuits. Coarse graining may be an effective approach to derive predictive phenomenological models for other ‘omics’ studies. PMID:25678603
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Advancing Cell Biology Through Proteomics in Space and Time (PROSPECTS)*
Lamond, Angus I.; Uhlen, Mathias; Horning, Stevan; Makarov, Alexander; Robinson, Carol V.; Serrano, Luis; Hartl, F. Ulrich; Baumeister, Wolfgang; Werenskiold, Anne Katrin; Andersen, Jens S.; Vorm, Ole; Linial, Michal; Aebersold, Ruedi; Mann, Matthias
2012-01-01
The term “proteomics” encompasses the large-scale detection and analysis of proteins and their post-translational modifications. Driven by major improvements in mass spectrometric instrumentation, methodology, and data analysis, the proteomics field has burgeoned in recent years. It now provides a range of sensitive and quantitative approaches for measuring protein structures and dynamics that promise to revolutionize our understanding of cell biology and molecular mechanisms in both human cells and model organisms. The Proteomics Specification in Time and Space (PROSPECTS) Network is a unique EU-funded project that brings together leading European research groups, spanning from instrumentation to biomedicine, in a collaborative five year initiative to develop new methods and applications for the functional analysis of cellular proteins. This special issue of Molecular and Cellular Proteomics presents 16 research papers reporting major recent progress by the PROSPECTS groups, including improvements to the resolution and sensitivity of the Orbitrap family of mass spectrometers, systematic detection of proteins using highly characterized antibody collections, and new methods for absolute as well as relative quantification of protein levels. Manuscripts in this issue exemplify approaches for performing quantitative measurements of cell proteomes and for studying their dynamic responses to perturbation, both during normal cellular responses and in disease mechanisms. Here we present a perspective on how the proteomics field is moving beyond simply identifying proteins with high sensitivity toward providing a powerful and versatile set of assay systems for characterizing proteome dynamics and thereby creating a new “third generation” proteomics strategy that offers an indispensible tool for cell biology and molecular medicine. PMID:22311636
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The accurate quantitation of proteins or peptides using Mass Spectrometry (MS) is gaining prominence in the biomedical research community as an alternative method for analyte measurement. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) investigators have been at the forefront in the promotion of reproducible MS techniques, through the development and application of standardized proteomic methods for protein quantitation on biologically relevant samples.
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Chang, Cheng; Xu, Kaikun; Guo, Chaoping; Wang, Jinxia; Yan, Qi; Zhang, Jian; He, Fuchu; Zhu, Yunping
2018-05-22
Compared with the numerous software tools developed for identification and quantification of -omics data, there remains a lack of suitable tools for both downstream analysis and data visualization. To help researchers better understand the biological meanings in their -omics data, we present an easy-to-use tool, named PANDA-view, for both statistical analysis and visualization of quantitative proteomics data and other -omics data. PANDA-view contains various kinds of analysis methods such as normalization, missing value imputation, statistical tests, clustering and principal component analysis, as well as the most commonly-used data visualization methods including an interactive volcano plot. Additionally, it provides user-friendly interfaces for protein-peptide-spectrum representation of the quantitative proteomics data. PANDA-view is freely available at https://sourceforge.net/projects/panda-view/. 1987ccpacer@163.com and zhuyunping@gmail.com. Supplementary data are available at Bioinformatics online.
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Ma, Danjun; Wang, Jiarui; Zhao, Yingchun; Lee, Wai-Nang Paul; Xiao, Jing; Go, Vay Liang W.; Wang, Qi; Recker, Robert; Xiao, Gary Guishan
2011-01-01
Objectives Novel quantitative proteomic approaches were used to study the effects of inhibition of glycogen phosphorylase on proteome and signaling pathways in MIA PaCa-2 pancreatic cancer cells. Methods We performed quantitative proteomic analysis in MIA PaCa-2 cancer cells treated with a stratified dose of CP-320626 (25 μM, 50 μM and 100 μM). The effect of metabolic inhibition on cellular protein turnover dynamics was also studied using the modified SILAC method (mSILAC). Results A total of twenty-two protein spots and four phosphoprotein spots were quantitatively analyzed. We found that dynamic expression of total proteins and phosphoproteins was significantly changed in MIA PaCa-2 cells treated with an incremental dose of CP-320626. Functional analyses suggested that most of the proteins differentially expressed were in the pathways of MAPK/ERK and TNF-α/NF-κB. Conclusions Signaling pathways and metabolic pathways share many common cofactors and substrates forming an extended metabolic network. The restriction of substrate through one pathway such as inhibition of glycogen phosphorylation induces pervasive metabolomic and proteomic changes manifested in protein synthesis, breakdown and post-translational modification of signaling molecules. Our results suggest that quantitative proteomic is an important approach to understand the interaction between metabolism and signaling pathways. PMID:22158071
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Solis, Nestor; Cain, Joel A; Cordwell, Stuart J
2016-01-01
Staphylococcus epidermidis is an opportunistic pathogen that is an emerging risk factor in hospitals worldwide and is often difficult to eradicate as virulent strains produce a protective biofilm matrix. We utilized cell shaving proteomics to profile surface-exposed proteins from two fully genome sequenced S. epidermidis strains: the avirulent, non-biofilm forming ATCC12228 and the virulent, strongly adherent biofilm forming ATCC35984 (RP62A). A false positive control strategy was employed to calculate the probabilities of proteins being truly surface-exposed. A total of 78 surface-exposed proteins were identified, of which only 19 proteins were common to ATCC12228 and RP62A, and which thus represents the core surfaceome. S. epidermidis RP62A displayed additional proteins involved in biofilm formation (cell wall-associated Bhp and intercellular adhesion protein IcaB), surface antigenicity, peptidoglycan biosynthesis and antibiotic resistance. We concurrently profiled whole cell proteomes of the two strains using iTRAQ quantitation and LC-MS/MS. A total of 1610 proteins were confidently identified (representing 64% of the theoretical S. epidermidis proteome). One hundred and ninety one proteins were differentially abundant between strains. Proteins associated with RP62A were clustered into functions including Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-mediated defense, sulfate assimilation, antibiotic resistance and biofilm formation. Validation of the sulfate assimilation and cysteine/methionine biosynthesis pathways showed RP62A contained elevated levels (~25% increase) of methionine that are likely linked to biofilm formation. Cell shaving and quantitative proteomics identified proteins associated with a biofilm-forming, virulent strain of S. epidermidis (RP62A). These proteins show RP62A maintains an active CRISPR-mediated defense, as well as heightened antibiotic resistance in comparison to a non-virulent, non-biofilm forming strain
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A proteomic approach to obesity and type 2 diabetes.
López-Villar, Elena; Martos-Moreno, Gabriel Á; Chowen, Julie A; Okada, Shigeru; Kopchick, John J; Argente, Jesús
2015-07-01
The incidence of obesity and type diabetes 2 has increased dramatically resulting in an increased interest in its biomedical relevance. However, the mechanisms that trigger the development of diabetes type 2 in obese patients remain largely unknown. Scientific, clinical and pharmaceutical communities are dedicating vast resources to unravel this issue by applying different omics tools. During the last decade, the advances in proteomic approaches and the Human Proteome Organization have opened and are opening a new door that may be helpful in the identification of patients at risk and to improve current therapies. Here, we briefly review some of the advances in our understanding of type 2 diabetes that have occurred through the application of proteomics. We also review, in detail, the current improvements in proteomic methodologies and new strategies that could be employed to further advance our understanding of this pathology. By applying these new proteomic advances, novel therapeutic and/or diagnostic protein targets will be discovered in the obesity/Type 2 diabetes area. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.
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Morris, Jeffrey S
2012-01-01
In recent years, developments in molecular biotechnology have led to the increased promise of detecting and validating biomarkers, or molecular markers that relate to various biological or medical outcomes. Proteomics, the direct study of proteins in biological samples, plays an important role in the biomarker discovery process. These technologies produce complex, high dimensional functional and image data that present many analytical challenges that must be addressed properly for effective comparative proteomics studies that can yield potential biomarkers. Specific challenges include experimental design, preprocessing, feature extraction, and statistical analysis accounting for the inherent multiple testing issues. This paper reviews various computational aspects of comparative proteomic studies, and summarizes contributions I along with numerous collaborators have made. First, there is an overview of comparative proteomics technologies, followed by a discussion of important experimental design and preprocessing issues that must be considered before statistical analysis can be done. Next, the two key approaches to analyzing proteomics data, feature extraction and functional modeling, are described. Feature extraction involves detection and quantification of discrete features like peaks or spots that theoretically correspond to different proteins in the sample. After an overview of the feature extraction approach, specific methods for mass spectrometry ( Cromwell ) and 2D gel electrophoresis ( Pinnacle ) are described. The functional modeling approach involves modeling the proteomic data in their entirety as functions or images. A general discussion of the approach is followed by the presentation of a specific method that can be applied, wavelet-based functional mixed models, and its extensions. All methods are illustrated by application to two example proteomic data sets, one from mass spectrometry and one from 2D gel electrophoresis. While the specific methods
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iTRAQ-Based Quantitative Proteomics of Developing and Ripening Muscadine Grape Berry
Kambiranda, Devaiah; Katam, Ramesh; Basha, Sheikh M.; Siebert, Shalom
2014-01-01
Grapes are among the widely cultivated fruit crops in the world. Grape berries like other nonclimacteric fruits undergo a complex set of dynamic, physical, physiological, and biochemical changes during ripening. Muscadine grapes are widely cultivated in the southern United States for fresh fruit and wine. To date, changes in the metabolites composition of muscadine grapes have been well documented; however, the molecular changes during berry development and ripening are not fully known. The aim of this study was to investigate changes in the berry proteome during ripening in muscadine grape cv. Noble. Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS was used to detect statistically significant changes in the berry proteome. A total of 674 proteins were detected, and 76 were differentially expressed across four time points in muscadine berry. Proteins obtained were further analyzed to provide information about its potential functions during ripening. Several proteins involved in abiotic and biotic stimuli and sucrose and hexose metabolism were upregulated during berry ripening. Quantitative real-time PCR analysis validated the protein expression results for nine proteins. Identification of vicilin-like antimicrobial peptides indicates additional disease tolerance proteins are present in muscadines for berry protection during ripening. The results provide new information for characterization and understanding muscadine berry proteome and grape ripening. PMID:24251720
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Lundquist, Peter K.; Poliakov, Anton; Bhuiyan, Nazmul H.; Zybailov, Boris; Sun, Qi; van Wijk, Klaas J.
2012-01-01
Plastoglobules (PGs) in chloroplasts are thylakoid-associated monolayer lipoprotein particles containing prenyl and neutral lipids and several dozen proteins mostly with unknown functions. An integrated view of the role of the PG is lacking. Here, we better define the PG proteome and provide a conceptual framework for further studies. The PG proteome from Arabidopsis (Arabidopsis thaliana) leaf chloroplasts was determined by mass spectrometry of isolated PGs and quantitative comparison with the proteomes of unfractionated leaves, thylakoids, and stroma. Scanning electron microscopy showed the purity and size distribution of the isolated PGs. Compared with previous PG proteome analyses, we excluded several proteins and identified six new PG proteins, including an M48 metallopeptidase and two Absence of bc1 complex (ABC1) atypical kinases, confirmed by immunoblotting. This refined PG proteome consisted of 30 proteins, including six ABC1 kinases and seven fibrillins together comprising more than 70% of the PG protein mass. Other fibrillins were located predominantly in the stroma or thylakoid and not in PGs; we discovered that this partitioning can be predicted by their isoelectric point and hydrophobicity. A genome-wide coexpression network for the PG genes was then constructed from mRNA expression data. This revealed a modular network with four distinct modules that each contained at least one ABC1K and/or fibrillin gene. Each module showed clear enrichment in specific functions, including chlorophyll degradation/senescence, isoprenoid biosynthesis, plastid proteolysis, and redox regulators and phosphoregulators of electron flow. We propose a new testable model for the PGs, in which sets of genes are associated with specific PG functions. PMID:22274653
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Loroch, Stefan; Schommartz, Tim; Brune, Wolfram; Zahedi, René Peiman; Sickmann, Albert
2015-05-01
Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein. To address this issue, we generated a highly sensitive workflow for combined LC-MS-based quantitative proteomics and phosphoproteomics by refining an ERLIC-based 2D phosphoproteomics workflow into an ERLIC-based 3D workflow covering the global proteome as well. The resulting 3D strategy was successfully used for an in-depth quantitative analysis of both, the proteome and the phosphoproteome of murine cytomegalovirus-infected mouse fibroblasts, a model system for host cell manipulation by a virus. In a 2-plex SILAC experiment with 150 μg of a tryptic digest per condition, the 3D strategy enabled the quantification of ~75% more proteins and even ~134% more peptides compared to the 2D strategy. Additionally, we could quantify ~50% more phosphoproteins by non-phosphorylated peptides, concurrently yielding insights into changes on the levels of protein expression and phosphorylation. Beside its sensitivity, our novel three-dimensional ERLIC-strategy has the potential for semi-automated sample processing rendering it a suitable future perspective for clinical, pre-clinical and biomedical research. Copyright © 2015. Published by Elsevier B.V.
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Liu, Zekun; Zhang, Qing-Bin; Bu, Chen; Wang, Dawei; Yu, Kai; Gan, Zhixue; Chang, Jianfeng; Cheng, Zhongyi; Liu, Zexian
2018-06-21
Stem-cell differentiation is a complex biological process controlled by a series of functional protein clusters and signaling transductions, especially metabolism-related pathways. Although previous studies have quantified the proteome and phosphoproteome for stem-cell differentiation, the investigation of acylation-mediated regulation is still absent. In this study, we quantitatively profiled the proteome, acetylome, and succinylome in pluripotent human embryonic stem cells (hESCs) and differentiated hepatocyte-like cells (HLCs). In total, 3843 proteins, 185 acetylation sites in 103 proteins, and 602 succinylation sites in 391 proteins were quantified. The quantitative proteome showed that in differentiated HLCs the TGF-β, JAK-STAT, and RAS signaling pathways were activated, whereas ECM-related processes such as sulfates and leucine degradation were depressed. Interestingly, it was observed that the acetylation and succinylation were more intensive in hESCs, whereas protein processing in endoplasmic reticulum and the carbon metabolic pathways were especially highly succinylated. Because the metabolism patterns in pluripotent hESCs and the differentiated HLCs were different, we proposed that the dynamic acylations, especially succinylation, might regulate the Warburg-like effect and TCA cycle during differentiation. Taken together, we systematically profiled the protein and acylation levels of regulation in pluripotent hESCs and differentiated HLCs, and the results indicated the important roles of acylation in pluripotency maintenance and differentiation.
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2013-01-01
Background Advances in DNA sequencing and proteomics have facilitated quantitative comparisons of snake venom composition. Most studies have employed one approach or the other. Here, both Illumina cDNA sequencing and LC/MS were used to compare the transcriptomes and proteomes of two pit vipers, Protobothrops flavoviridis and Ovophis okinavensis, which differ greatly in their biology. Results Sequencing of venom gland cDNA produced 104,830 transcripts. The Protobothrops transcriptome contained transcripts for 103 venom-related proteins, while the Ovophis transcriptome contained 95. In both, transcript abundances spanned six orders of magnitude. Mass spectrometry identified peptides from 100% of transcripts that occurred at higher than contaminant (e.g. human keratin) levels, including a number of proteins never before sequenced from snakes. These transcriptomes reveal fundamentally different envenomation strategies. Adult Protobothrops venom promotes hemorrhage, hypotension, incoagulable blood, and prey digestion, consistent with mammalian predation. Ovophis venom composition is less readily interpreted, owing to insufficient pharmacological data for venom serine and metalloproteases, which comprise more than 97.3% of Ovophis transcripts, but only 38.0% of Protobothrops transcripts. Ovophis venom apparently represents a hybrid strategy optimized for frogs and small mammals. Conclusions This study illustrates the power of cDNA sequencing combined with MS profiling. The former quantifies transcript composition, allowing detection of novel proteins, but cannot indicate which proteins are actually secreted, as does MS. We show, for the first time, that transcript and peptide abundances are correlated. This means that MS can be used for quantitative, non-invasive venom profiling, which will be beneficial for studies of endangered species. PMID:24224955
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Systematic Proteomic Approach to Characterize the Impacts of ...
Chemical interactions have posed a big challenge in toxicity characterization and human health risk assessment of environmental mixtures. To characterize the impacts of chemical interactions on protein and cytotoxicity responses to environmental mixtures, we established a systems biology approach integrating proteomics, bioinformatics, statistics, and computational toxicology to measure expression or phosphorylation levels of 21 critical toxicity pathway regulators and 445 downstream proteins in human BEAS-28 cells treated with 4 concentrations of nickel, 2 concentrations each of cadmium and chromium, as well as 12 defined binary and 8 defined ternary mixtures of these metals in vitro. Multivariate statistical analysis and mathematical modeling of the metal-mediated proteomic response patterns showed a high correlation between changes in protein expression or phosphorylation and cellular toxic responses to both individual metals and metal mixtures. Of the identified correlated proteins, only a small set of proteins including HIF-1a is likely to be responsible for selective cytotoxic responses to different metals and metals mixtures. Furthermore, support vector machine learning was utilized to computationally predict protein responses to uncharacterized metal mixtures using experimentally generated protein response profiles corresponding to known metal mixtures. This study provides a novel proteomic approach for characterization and prediction of toxicities of
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High-coverage quantitative proteomics using amine-specific isotopic labeling.
Melanson, Jeremy E; Avery, Steven L; Pinto, Devanand M
2006-08-01
Peptide dimethylation with isotopically coded formaldehydes was evaluated as a potential alternative to techniques such as the iTRAQ method for comparative proteomics. The isotopic labeling strategy and custom-designed protein quantitation software were tested using protein standards and then applied to measure proteins levels associated with Alzheimer's disease (AD). The method provided high accuracy (10% error), precision (14% RSD) and coverage (70%) when applied to the analysis of a standard solution of BSA by LC-MS/MS. The technique was then applied to measure protein abundance levels in brain tissue afflicted with AD relative to normal brain tissue. 2-D LC-MS analysis identified 548 unique proteins (p<0.05). Of these, 349 were quantified with two or more peptides that met the statistical criteria used in this study. Several classes of proteins exhibited significant changes in abundance. For example, elevated levels of antioxidant proteins and decreased levels of mitochondrial electron transport proteins were observed. The results demonstrate the utility of the labeling method for high-throughput quantitative analysis.
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Shah, Anup D; Inder, Kerry L; Shah, Alok K; Cristino, Alexandre S; McKie, Arthur B; Gabra, Hani; Davis, Melissa J; Hill, Michelle M
2016-10-07
Lipid rafts are dynamic membrane microdomains that orchestrate molecular interactions and are implicated in cancer development. To understand the functions of lipid rafts in cancer, we performed an integrated analysis of quantitative lipid raft proteomics data sets modeling progression in breast cancer, melanoma, and renal cell carcinoma. This analysis revealed that cancer development is associated with increased membrane raft-cytoskeleton interactions, with ∼40% of elevated lipid raft proteins being cytoskeletal components. Previous studies suggest a potential functional role for the raft-cytoskeleton in the action of the putative tumor suppressors PTRF/Cavin-1 and Merlin. To extend the observation, we examined lipid raft proteome modulation by an unrelated tumor suppressor opioid binding protein cell-adhesion molecule (OPCML) in ovarian cancer SKOV3 cells. In agreement with the other model systems, quantitative proteomics revealed that 39% of OPCML-depleted lipid raft proteins are cytoskeletal components, with microfilaments and intermediate filaments specifically down-regulated. Furthermore, protein-protein interaction network and simulation analysis showed significantly higher interactions among cancer raft proteins compared with general human raft proteins. Collectively, these results suggest increased cytoskeleton-mediated stabilization of lipid raft domains with greater molecular interactions as a common, functional, and reversible feature of cancer cells.
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Sadewasser, Anne; Paki, Katharina; Eichelbaum, Katrin; Bogdanow, Boris; Saenger, Sandra; Budt, Matthias; Lesch, Markus; Hinz, Klaus-Peter; Herrmann, Andreas; Meyer, Thomas F; Karlas, Alexander; Selbach, Matthias; Wolff, Thorsten
2017-05-01
Influenza A virus (IAV) infections are a major cause for respiratory disease in humans, which affects all age groups and contributes substantially to global morbidity and mortality. IAV have a large natural host reservoir in avian species. However, many avian IAV strains lack adaptation to other hosts and hardly propagate in humans. While seasonal or pandemic IAV strains replicate efficiently in permissive human cells, many avian IAV cause abortive nonproductive infections in these hosts despite successful cell entry. However, the precise reasons for these differential outcomes are poorly defined. We hypothesized that the distinct course of an IAV infection with a given virus strain is determined by the differential interplay between specific host and viral factors. By using Spike-in SILAC mass spectrometry-based quantitative proteomics we characterized sets of cellular factors whose abundance is specifically up- or downregulated in the course of permissive versus nonpermissive IAV infection, respectively. This approach allowed for the definition and quantitative comparison of about 3500 proteins in human lung epithelial cells in response to seasonal or low-pathogenic avian H3N2 IAV. Many identified proteins were similarly regulated by both virus strains, but also 16 candidates with distinct changes in permissive versus nonpermissive infection were found. RNAi-mediated knockdown of these differentially regulated host factors identified Vpr binding protein (VprBP) as proviral host factor because its downregulation inhibited efficient propagation of seasonal IAV whereas overexpression increased viral replication of both seasonal and avian IAV. These results not only show that there are similar differences in the overall changes during permissive and nonpermissive influenza virus infections, but also provide a basis to evaluate VprBP as novel anti-IAV drug target. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Sadewasser, Anne; Paki, Katharina; Eichelbaum, Katrin; Bogdanow, Boris; Saenger, Sandra; Budt, Matthias; Lesch, Markus; Hinz, Klaus-Peter; Herrmann, Andreas; Meyer, Thomas F.; Karlas, Alexander; Selbach, Matthias; Wolff, Thorsten
2017-01-01
Influenza A virus (IAV) infections are a major cause for respiratory disease in humans, which affects all age groups and contributes substantially to global morbidity and mortality. IAV have a large natural host reservoir in avian species. However, many avian IAV strains lack adaptation to other hosts and hardly propagate in humans. While seasonal or pandemic IAV strains replicate efficiently in permissive human cells, many avian IAV cause abortive nonproductive infections in these hosts despite successful cell entry. However, the precise reasons for these differential outcomes are poorly defined. We hypothesized that the distinct course of an IAV infection with a given virus strain is determined by the differential interplay between specific host and viral factors. By using Spike-in SILAC mass spectrometry-based quantitative proteomics we characterized sets of cellular factors whose abundance is specifically up- or downregulated in the course of permissive versus nonpermissive IAV infection, respectively. This approach allowed for the definition and quantitative comparison of about 3500 proteins in human lung epithelial cells in response to seasonal or low-pathogenic avian H3N2 IAV. Many identified proteins were similarly regulated by both virus strains, but also 16 candidates with distinct changes in permissive versus nonpermissive infection were found. RNAi-mediated knockdown of these differentially regulated host factors identified Vpr binding protein (VprBP) as proviral host factor because its downregulation inhibited efficient propagation of seasonal IAV whereas overexpression increased viral replication of both seasonal and avian IAV. These results not only show that there are similar differences in the overall changes during permissive and nonpermissive influenza virus infections, but also provide a basis to evaluate VprBP as novel anti-IAV drug target. PMID:28289176
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Detergents: Friends not foes for high-performance membrane proteomics toward precision medicine.
Zhang, Xi
2017-02-01
Precision medicine, particularly therapeutics, emphasizes the atomic-precise, dynamic, and systems visualization of human membrane proteins and their endogenous modifiers. For years, bottom-up proteomics has grappled with removing and avoiding detergents, yet faltered at the therapeutic-pivotal membrane proteins, which have been tackled by classical approaches and are known for decades refractory to single-phase aqueous or organic denaturants. Hydrophobicity and aggregation commonly challenge tissue and cell lysates, biofluids, and enriched samples. Frequently, expected membrane proteins and peptides are not identified by shotgun bottom-up proteomics, let alone robust quantitation. This review argues the cause of this proteomic crisis is not detergents per se, but the choice of detergents. Recently, inclusion of compatible detergents for membrane protein extraction and digestion has revealed stark improvements in both quantitative and structural proteomics. This review analyzes detergent properties behind recent proteomic advances, and proposes that rational use of detergents may reconcile outstanding membrane proteomics dilemmas, enabling ultradeep coverage and minimal artifacts for robust protein and endogenous PTM measurements. The simplicity of detergent tools confers bottom-up membrane proteomics the sophistication toward precision medicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Gao, Kun; Deng, Xiang-Yuan; Shang, Meng-Ke; Qin, Guang-Xing; Hou, Cheng-Xiang; Guo, Xi-Jie
2017-01-30
Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes them to death, which negatively affects the sericulture industry. In order to determine the midgut response at the protein levels to the virus infection, differential proteomes of the silkworm midgut responsive to BmCPV infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). 193, 408, 189 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the midgut of BmCPV-infected and control larvae at 24, 48, 72h post infection (hpi) respectively. KEGG enrichment analysis showed that Oxidative phosphorylation, amyotrophic lateral sclerosis, Toll-like receptor signaling pathway, steroid hormone biosynthesis were the significant pathways (Q value≤0.05) both at 24 and 48hpi. qRT-PCR was used to further verify gene transcription of 30 DEPs from iTRAQ, showing that the regulations of 24 genes at the transcript level were consistent with those at the proteomic level. Moreover, the cluster analysis of the three time groups showed that there were seven co-regulated DEPs including BGIBMGA002620-PA, which was a putative p62/sequestosome-1 protein in silkworm. It was upregulated at both the mRNA level and the proteomic level and may play an important role in regulating the autophagy and apoptosis (especially apoptosis) induced by BmCPV infection. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection. The domesticated silkworm, Bombyx mori, is renowned for silk production as well as being a traditional lepidopteron model insect served as a subject for morphological, genetic, physiological, and developmental studies. Bombyx mori cytoplasmic polyhedrosis
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Marino, Daniel; Ariz, Idoia; Lasa, Berta; Santamaría, Enrique; Fernández-Irigoyen, Joaquín; González-Murua, Carmen; Aparicio Tejo, Pedro M.
2016-01-01
Accessing different nitrogen (N) sources involves a profound adaptation of plant metabolism. In this study, a quantitative proteomic approach was used to further understand how the model plant Arabidopsis thaliana adjusts to different N sources when grown exclusively under nitrate or ammonium nutrition. Proteome data evidenced that glucosinolate metabolism was differentially regulated by the N source and that both TGG1 and TGG2 myrosinases were more abundant under ammonium nutrition, which is generally considered to be a stressful situation. Moreover, Arabidopsis plants displayed glucosinolate accumulation and induced myrosinase activity under ammonium nutrition. Interestingly, these results were also confirmed in the economically important crop broccoli (Brassica oleracea var. italica). Moreover, these metabolic changes were correlated in Arabidopsis with the differential expression of genes from the aliphatic glucosinolate metabolic pathway. This study underlines the importance of nitrogen nutrition and the potential of using ammonium as the N source in order to stimulate glucosinolate metabolism, which may have important applications not only in terms of reducing pesticide use, but also for increasing plants’ nutritional value. PMID:27085186
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Boja, Emily S; Rodriguez, Henry
2012-04-01
Traditional shotgun proteomics used to detect a mixture of hundreds to thousands of proteins through mass spectrometric analysis, has been the standard approach in research to profile protein content in a biological sample which could lead to the discovery of new (and all) protein candidates with diagnostic, prognostic, and therapeutic values. In practice, this approach requires significant resources and time, and does not necessarily represent the goal of the researcher who would rather study a subset of such discovered proteins (including their variations or posttranslational modifications) under different biological conditions. In this context, targeted proteomics is playing an increasingly important role in the accurate measurement of protein targets in biological samples in the hope of elucidating the molecular mechanism of cellular function via the understanding of intricate protein networks and pathways. One such (targeted) approach, selected reaction monitoring (or multiple reaction monitoring) mass spectrometry (MRM-MS), offers the capability of measuring multiple proteins with higher sensitivity and throughput than shotgun proteomics. Developing and validating MRM-MS-based assays, however, is an extensive and iterative process, requiring a coordinated and collaborative effort by the scientific community through the sharing of publicly accessible data and datasets, bioinformatic tools, standard operating procedures, and well characterized reagents. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Large-scale label-free quantitative proteomics of the pea aphid-Buchnera symbiosis.
Poliakov, Anton; Russell, Calum W; Ponnala, Lalit; Hoops, Harold J; Sun, Qi; Douglas, Angela E; van Wijk, Klaas J
2011-06-01
Many insects are nutritionally dependent on symbiotic microorganisms that have tiny genomes and are housed in specialized host cells called bacteriocytes. The obligate symbiosis between the pea aphid Acyrthosiphon pisum and the γ-proteobacterium Buchnera aphidicola (only 584 predicted proteins) is particularly amenable for molecular analysis because the genomes of both partners have been sequenced. To better define the symbiotic relationship between this aphid and Buchnera, we used large-scale, high accuracy tandem mass spectrometry (nanoLC-LTQ-Orbtrap) to identify aphid and Buchnera proteins in the whole aphid body, purified bacteriocytes, isolated Buchnera cells and the residual bacteriocyte fraction. More than 1900 aphid and 400 Buchnera proteins were identified. All enzymes in amino acid metabolism annotated in the Buchnera genome were detected, reflecting the high (68%) coverage of the proteome and supporting the core function of Buchnera in the aphid symbiosis. Transporters mediating the transport of predicted metabolites were present in the bacteriocyte. Label-free spectral counting combined with hierarchical clustering, allowed to define the quantitative distribution of a subset of these proteins across both symbiotic partners, yielding no evidence for the selective transfer of protein among the partners in either direction. This is the first quantitative proteome analysis of bacteriocyte symbiosis, providing a wealth of information about molecular function of both the host cell and bacterial symbiont.
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Lee, Chang-Ro; Lee, Jung Hun; Park, Kwang Seung; Jeong, Byeong Chul; Lee, Sang Hee
2015-01-01
The increase of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) poses a worldwide and serious health threat. Although new antibiotics, such as daptomycin and linezolid, have been developed for the treatment of infections of Gram-positive pathogens, the emergence of daptomycin-resistant and linezolid-resistant strains during therapy has now increased clinical treatment failures. In the past few years, studies using quantitative proteomic methods have provided a considerable progress in understanding antibiotic resistance mechanisms. In this review, to understand the resistance mechanisms to four clinically important antibiotics (methicillin, vancomycin, linezolid, and daptomycin) used in the treatment of Gram-positive pathogens, we summarize recent advances in studies on resistance mechanisms using quantitative proteomic methods, and also examine proteins playing an important role in the bacterial mechanisms of resistance to the four antibiotics. Proteomic researches can identify proteins whose expression levels are changed in the resistance mechanism to only one antibiotic, such as LiaH in daptomycin resistance and PrsA in vancomycin resistance, and many proteins simultaneously involved in resistance mechanisms to various antibiotics. Most of resistance-related proteins, which are simultaneously associated with resistance mechanisms to several antibiotics, play important roles in regulating bacterial envelope biogenesis, or compensating for the fitness cost of antibiotic resistance. Therefore, proteomic data confirm that antibiotic resistance requires the fitness cost and the bacterial envelope is an important factor in antibiotic resistance. PMID:26322035
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2015-01-01
Atropine, a muscarinic antagonist, is known to inhibit myopia progression in several animal models and humans. However, the mode of action is not established yet. In this study, we compared quantitative iTRAQ proteomic analysis in the retinas collected from control and lens-induced myopic (LIM) mouse eyes treated with atropine. The myopic group received a (−15D) spectacle lens over the right eye on postnatal day 10 with or without atropine eye drops starting on postnatal day 24. Axial length was measured by optical low coherence interferometry (OLCI), AC-Master, and refraction was measured by automated infrared photorefractor at postnatal 24, 38, and 52 days. Retinal tissue samples were pooled from six eyes for each group. The experiments were repeated twice, and technical replicates were also performed for liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. MetaCore was used to perform protein profiling for pathway analysis. We identified a total of 3882 unique proteins with <1% FDR by analyzing the samples in replicates for two independent experiments. This is the largest number of mouse retina proteome reported to date. Thirty proteins were found to be up-regulated (ratio for myopia/control > global mean ratio + 1 standard deviation), and 28 proteins were down-regulated (ratio for myopia/control < global mean ratio - 1 standard deviation) in myopic eyes as compared with control retinas. Pathway analysis using MetaCore revealed regulation of γ-aminobutyric acid (GABA) levels in the myopic eyes. Detailed analysis of the quantitative proteomics data showed that the levels of GABA transporter 1 (GAT-1) were elevated in myopic retina and significantly reduced after atropine treatment. These results were further validated with immunohistochemistry and Western blot analysis. In conclusion, this study provides a comprehensive quantitative proteomic analysis of atropine-treated mouse retina and suggests the involvement of GABAergic signaling in the
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iTRAQ-Based Quantitative Proteomic Analysis of the Initiation of Head Regeneration in Planarians.
Geng, Xiaofang; Wang, Gaiping; Qin, Yanli; Zang, Xiayan; Li, Pengfei; Geng, Zhi; Xue, Deming; Dong, Zimei; Ma, Kexue; Chen, Guangwen; Xu, Cunshuan
2015-01-01
The planarian Dugesia japonica has amazing ability to regenerate a head from the anterior ends of the amputated stump with maintenance of the original anterior-posterior polarity. Although planarians present an attractive system for molecular investigation of regeneration and research has focused on clarifying the molecular mechanism of regeneration initiation in planarians at transcriptional level, but the initiation mechanism of planarian head regeneration (PHR) remains unclear at the protein level. Here, a global analysis of proteome dynamics during the early stage of PHR was performed using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics strategy, and our data are available via ProteomeXchange with identifier PXD002100. The results showed that 162 proteins were differentially expressed at 2 h and 6 h following amputation. Furthermore, the analysis of expression patterns and functional enrichment of the differentially expressed proteins showed that proteins involved in muscle contraction, oxidation reduction and protein synthesis were up-regulated in the initiation of PHR. Moreover, ingenuity pathway analysis showed that predominant signaling pathways such as ILK, calcium, EIF2 and mTOR signaling which were associated with cell migration, cell proliferation and protein synthesis were likely to be involved in the initiation of PHR. The results for the first time demonstrated that muscle contraction and ILK signaling might played important roles in the initiation of PHR at the global protein level. The findings of this research provide a molecular basis for further unraveling the mechanism of head regeneration initiation in planarians.
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Affinity Proteomics for Fast, Sensitive, Quantitative Analysis of Proteins in Plasma.
O'Grady, John P; Meyer, Kevin W; Poe, Derrick N
2017-01-01
The improving efficacy of many biological therapeutics and identification of low-level biomarkers are driving the analytical proteomics community to deal with extremely high levels of sample complexity relative to their analytes. Many protein quantitation and biomarker validation procedures utilize an immunoaffinity enrichment step to purify the sample and maximize the sensitivity of the corresponding liquid chromatography tandem mass spectrometry measurements. In order to generate surrogate peptides with better mass spectrometric properties, protein enrichment is followed by a proteolytic cleavage step. This is often a time-consuming multistep process. Presented here is a workflow which enables rapid protein enrichment and proteolytic cleavage to be performed in a single, easy-to-use reactor. Using this strategy Klotho, a low-abundance biomarker found in plasma, can be accurately quantitated using a protocol that takes under 5 h from start to finish.
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NCI Launches Proteomics Assay Portal | Office of Cancer Clinical Proteomics Research
In a paper recently published by the journal Nature Methods, Investigators from the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (NCI-CPTAC) announced the launch of a proteomics Assay Portal for multiple reaction monitoring-mass spectrometry (MRM-MS) assays. This community web-based repository for well-characterized quantitative proteomic assays currently consists of 456 unique peptide assays to 282 unique proteins and ser
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Salivary biomarker development using genomic, proteomic and metabolomic approaches
2012-01-01
The use of saliva as a diagnostic sample provides a non-invasive, cost-efficient method of sample collection for disease screening without the need for highly trained professionals. Saliva collection is far more practical and safe compared with invasive methods of sample collection, because of the infection risk from contaminated needles during, for example, blood sampling. Furthermore, the use of saliva could increase the availability of accurate diagnostics for remote and impoverished regions. However, the development of salivary diagnostics has required technical innovation to allow stabilization and detection of analytes in the complex molecular mixture that is saliva. The recent development of cost-effective room temperature analyte stabilization methods, nucleic acid pre-amplification techniques and direct saliva transcriptomic analysis have allowed accurate detection and quantification of transcripts found in saliva. Novel protein stabilization methods have also facilitated improved proteomic analyses. Although candidate biomarkers have been discovered using epigenetic, transcriptomic, proteomic and metabolomic approaches, transcriptomic analyses have so far achieved the most progress in terms of sensitivity and specificity, and progress towards clinical implementation. Here, we review recent developments in salivary diagnostics that have been accomplished using genomic, transcriptomic, proteomic and metabolomic approaches. PMID:23114182
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Gao, Yan; Lim, Teck Kwang; Lin, Qingsong; Li, Sam Fong Yau
2016-04-29
Cypermethrin (CYP) is one of the most widely used pesticides in large scale for agricultural and domestic purpose and the residue often seriously affects aquatic system. Environmental pollutant-induced protein changes in organisms could be detected by proteomics, leading to discovery of potential biomarkers and understanding of mode of action. While proteomics investigations of CYP stress in some animal models have been well studied, few reports about the effects of exposure to CYP on algae proteome were published. To determine CYP effect in algae, the impact of various dosages (0.001μg/L, 0.01μg/L and 1μg/L) of CYP on green algae Chlorella vulgaris for 24h and 96h was investigated by using iTRAQ quantitative proteomics technique. A total of 162 and 198 proteins were significantly altered after CYP exposure for 24h and 96h, respectively. Overview of iTRAQ results indicated that the influence of CYP on algae protein might be dosage-dependent. Functional analysis of differentially expressed proteins showed that CYP could induce protein alterations related to photosynthesis, stress responses and carbohydrate metabolism. This study provides a comprehensive view of complex mode of action of algae under CYP stress and highlights several potential biomarkers for further investigation of pesticide-exposed plant and algae. Copyright © 2016 Elsevier B.V. All rights reserved.
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Dejung, Mario; Subota, Ines; Bucerius, Ferdinand; Dindar, Gülcin; Freiwald, Anja; Engstler, Markus; Boshart, Michael; Butter, Falk; Janzen, Christian J.
2016-01-01
Developmental differentiation is a universal biological process that allows cells to adapt to different environments to perform specific functions. African trypanosomes progress through a tightly regulated life cycle in order to survive in different host environments when they shuttle between an insect vector and a vertebrate host. Transcriptomics has been useful to gain insight into RNA changes during stage transitions; however, RNA levels are only a moderate proxy for protein abundance in trypanosomes. We quantified 4270 protein groups during stage differentiation from the mammalian-infective to the insect form and provide classification for their expression profiles during development. Our label-free quantitative proteomics study revealed previously unknown components of the differentiation machinery that are involved in essential biological processes such as signaling, posttranslational protein modifications, trafficking and nuclear transport. Furthermore, guided by our proteomic survey, we identified the cause of the previously observed differentiation impairment in the histone methyltransferase DOT1B knock-out strain as it is required for accurate karyokinesis in the first cell division during differentiation. This epigenetic regulator is likely involved in essential chromatin restructuring during developmental differentiation, which might also be important for differentiation in higher eukaryotic cells. Our proteome dataset will serve as a resource for detailed investigations of cell differentiation to shed more light on the molecular mechanisms of this process in trypanosomes and other eukaryotes. PMID:26910529
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Yi, Zhou; Manil-Ségalen, Marion; Sago, Laila; Glatigny, Annie; Redeker, Virginie; Legouis, Renaud; Mucchielli-Giorgi, Marie-Hélène
2016-05-06
Affinity purifications followed by mass spectrometric analysis are used to identify protein-protein interactions. Because quantitative proteomic data are noisy, it is necessary to develop statistical methods to eliminate false-positives and identify true partners. We present here a novel approach for filtering false interactors, named "SAFER" for mass Spectrometry data Analysis by Filtering of Experimental Replicates, which is based on the reproducibility of the replicates and the fold-change of the protein intensities between bait and control. To identify regulators or targets of autophagy, we characterized the interactors of LGG1, a ubiquitin-like protein involved in autophagosome formation in C. elegans. LGG-1 partners were purified by affinity, analyzed by nanoLC-MS/MS mass spectrometry, and quantified by a label-free proteomic approach based on the mass spectrometric signal intensity of peptide precursor ions. Because the selection of confident interactions depends on the method used for statistical analysis, we compared SAFER with several statistical tests and different scoring algorithms on this set of data. We show that SAFER recovers high-confidence interactors that have been ignored by the other methods and identified new candidates involved in the autophagy process. We further validated our method on a public data set and conclude that SAFER notably improves the identification of protein interactors.
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Proteomic approaches to study the pig intestinal system.
Soler, Laura; Niewold, Theo A; Moreno, Ángela; Garrido, Juan Jose
2014-03-01
One of the major challenges in pig production is managing digestive health to maximize feed conversion and growth rates, but also to minimize treatment costs and to warrant public health. There is a great interest in the development of useful tools for intestinal health monitoring and the investigation of possible prophylactic/ therapeutic intervention pathways. A great variety of in vivo and in vitro intestinal models of study have been developed in the recent years. The understanding of such a complex system as the intestinal system (IS), and the study of its physiology and pathology is not an easy task. Analysis of such a complex system requires the use of systems biology techniques, like proteomics. However, for a correct interpretation of results and to maximize analysis performance, a careful selection of the IS model of study and proteomic platform is required. The study of the IS system is especially important in the pig, a species whose farming requires a very careful management of husbandry procedures regarding feeding and nutrition. The incorrect management of the pig digestive system leads directly to economic losses related suboptimal growth and feed utilization and/or the appearance of intestinal infections, in particular diarrhea. Furthermore, this species is the most suitable experimental model for human IS studies. Proteomics has risen as one of the most promising approaches to study the pig IS. In this review, we describe the most useful models of IS research in porcine and the different proteomic platforms available. An overview of the recent findings in pig IS proteomics is also provided.
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Halobacterium salinarum NRC-1 PeptideAtlas: strategies for targeted proteomics
Van, Phu T.; Schmid, Amy K.; King, Nichole L.; Kaur, Amardeep; Pan, Min; Whitehead, Kenia; Koide, Tie; Facciotti, Marc T.; Goo, Young-Ah; Deutsch, Eric W.; Reiss, David J.; Mallick, Parag; Baliga, Nitin S.
2009-01-01
The relatively small numbers of proteins and fewer possible posttranslational modifications in microbes provides a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a Peptide Atlas (PA) for 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636,000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has helped highlight plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics. PMID:18652504
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Jorge, Inmaculada; Navarro, Pedro; Martínez-Acedo, Pablo; Núñez, Estefanía; Serrano, Horacio; Alfranca, Arántzazu; Redondo, Juan Miguel; Vázquez, Jesús
2009-01-01
Statistical models for the analysis of protein expression changes by stable isotope labeling are still poorly developed, particularly for data obtained by 16O/18O labeling. Besides large scale test experiments to validate the null hypothesis are lacking. Although the study of mechanisms underlying biological actions promoted by vascular endothelial growth factor (VEGF) on endothelial cells is of considerable interest, quantitative proteomics studies on this subject are scarce and have been performed after exposing cells to the factor for long periods of time. In this work we present the largest quantitative proteomics study to date on the short term effects of VEGF on human umbilical vein endothelial cells by 18O/16O labeling. Current statistical models based on normality and variance homogeneity were found unsuitable to describe the null hypothesis in a large scale test experiment performed on these cells, producing false expression changes. A random effects model was developed including four different sources of variance at the spectrum-fitting, scan, peptide, and protein levels. With the new model the number of outliers at scan and peptide levels was negligible in three large scale experiments, and only one false protein expression change was observed in the test experiment among more than 1000 proteins. The new model allowed the detection of significant protein expression changes upon VEGF stimulation for 4 and 8 h. The consistency of the changes observed at 4 h was confirmed by a replica at a smaller scale and further validated by Western blot analysis of some proteins. Most of the observed changes have not been described previously and are consistent with a pattern of protein expression that dynamically changes over time following the evolution of the angiogenic response. With this statistical model the 18O labeling approach emerges as a very promising and robust alternative to perform quantitative proteomics studies at a depth of several thousand proteins
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Deutsch, Eric W.; Mendoza, Luis; Shteynberg, David; Slagel, Joseph; Sun, Zhi; Moritz, Robert L.
2015-01-01
Democratization of genomics technologies has enabled the rapid determination of genotypes. More recently the democratization of comprehensive proteomics technologies is enabling the determination of the cellular phenotype and the molecular events that define its dynamic state. Core proteomic technologies include mass spectrometry to define protein sequence, protein:protein interactions, and protein post-translational modifications. Key enabling technologies for proteomics are bioinformatic pipelines to identify, quantitate, and summarize these events. The Trans-Proteomics Pipeline (TPP) is a robust open-source standardized data processing pipeline for large-scale reproducible quantitative mass spectrometry proteomics. It supports all major operating systems and instrument vendors via open data formats. Here we provide a review of the overall proteomics workflow supported by the TPP, its major tools, and how it can be used in its various modes from desktop to cloud computing. We describe new features for the TPP, including data visualization functionality. We conclude by describing some common perils that affect the analysis of tandem mass spectrometry datasets, as well as some major upcoming features. PMID:25631240
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Deutsch, Eric W; Mendoza, Luis; Shteynberg, David; Slagel, Joseph; Sun, Zhi; Moritz, Robert L
2015-08-01
Democratization of genomics technologies has enabled the rapid determination of genotypes. More recently the democratization of comprehensive proteomics technologies is enabling the determination of the cellular phenotype and the molecular events that define its dynamic state. Core proteomic technologies include MS to define protein sequence, protein:protein interactions, and protein PTMs. Key enabling technologies for proteomics are bioinformatic pipelines to identify, quantitate, and summarize these events. The Trans-Proteomics Pipeline (TPP) is a robust open-source standardized data processing pipeline for large-scale reproducible quantitative MS proteomics. It supports all major operating systems and instrument vendors via open data formats. Here, we provide a review of the overall proteomics workflow supported by the TPP, its major tools, and how it can be used in its various modes from desktop to cloud computing. We describe new features for the TPP, including data visualization functionality. We conclude by describing some common perils that affect the analysis of MS/MS datasets, as well as some major upcoming features. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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NCI's Proteome Characterization Centers Announced | Office of Cancer Clinical Proteomics Research
The National Cancer Institute (NCI), part of the National Institutes of Health, announces the launch of a Clinical Proteomic Tumor Analysis Consortium (CPTAC). CPTAC is a comprehensive, coordinated team effort to accelerate the understanding of the molecular basis of cancer through the application of robust, quantitative, proteomic technologies and workflows.
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Label free quantitative proteomics analysis on the cisplatin resistance in ovarian cancer cells.
Wang, F; Zhu, Y; Fang, S; Li, S; Liu, S
2017-05-20
Quantitative proteomics has been made great progress in recent years. Label free quantitative proteomics analysis based on the mass spectrometry is widely used. Using this technique, we determined the differentially expressed proteins in the cisplatin-sensitive ovarian cancer cells COC1 and cisplatin-resistant cells COC1/DDP before and after the application of cisplatin. Using the GO analysis, we classified those proteins into different subgroups bases on their cellular component, biological process, and molecular function. We also used KEGG pathway analysis to determine the key signal pathways that those proteins were involved in. There are 710 differential proteins between COC1 and COC1/DDP cells, 783 between COC1 and COC1/DDP cells treated with cisplatin, 917 between the COC1/DDP cells and COC1/DDP cells treated with LaCl3, 775 between COC1/DDP cells treated with cisplatin and COC1/DDP cells treated with cisplatin and LaCl3. Among the same 411 differentially expressed proteins in cisplatin-sensitive COC1 cells and cisplain-resistant COC1/DDP cells before and after cisplatin treatment, 14% of them were localized on the cell membrane. According to the KEGG results, differentially expressed proteins were classified into 21 groups. The most abundant proteins were involved in spliceosome. This study lays a foundation for deciphering the mechanism for drug resistance in ovarian tumor.
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Stauch, Kelly L; Purnell, Phillip R; Fox, Howard S
2014-05-02
Synaptic mitochondria are essential for maintaining calcium homeostasis and producing ATP, processes vital for neuronal integrity and synaptic transmission. Synaptic mitochondria exhibit increased oxidative damage during aging and are more vulnerable to calcium insult than nonsynaptic mitochondria. Why synaptic mitochondria are specifically more susceptible to cumulative damage remains to be determined. In this study, the generation of a super-SILAC mix that served as an appropriate internal standard for mouse brain mitochondria mass spectrometry based analysis allowed for the quantification of the proteomic differences between synaptic and nonsynaptic mitochondria isolated from 10-month-old mice. We identified a total of 2260 common proteins between synaptic and nonsynaptic mitochondria of which 1629 were annotated as mitochondrial. Quantitative proteomic analysis of the proteins common between synaptic and nonsynaptic mitochondria revealed significant differential expression of 522 proteins involved in several pathways including oxidative phosphorylation, mitochondrial fission/fusion, calcium transport, and mitochondrial DNA replication and maintenance. In comparison to nonsynaptic mitochondria, synaptic mitochondria exhibited increased age-associated mitochondrial DNA deletions and decreased bioenergetic function. These findings provide insights into synaptic mitochondrial susceptibility to damage.
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2015-01-01
Synaptic mitochondria are essential for maintaining calcium homeostasis and producing ATP, processes vital for neuronal integrity and synaptic transmission. Synaptic mitochondria exhibit increased oxidative damage during aging and are more vulnerable to calcium insult than nonsynaptic mitochondria. Why synaptic mitochondria are specifically more susceptible to cumulative damage remains to be determined. In this study, the generation of a super-SILAC mix that served as an appropriate internal standard for mouse brain mitochondria mass spectrometry based analysis allowed for the quantification of the proteomic differences between synaptic and nonsynaptic mitochondria isolated from 10-month-old mice. We identified a total of 2260 common proteins between synaptic and nonsynaptic mitochondria of which 1629 were annotated as mitochondrial. Quantitative proteomic analysis of the proteins common between synaptic and nonsynaptic mitochondria revealed significant differential expression of 522 proteins involved in several pathways including oxidative phosphorylation, mitochondrial fission/fusion, calcium transport, and mitochondrial DNA replication and maintenance. In comparison to nonsynaptic mitochondria, synaptic mitochondria exhibited increased age-associated mitochondrial DNA deletions and decreased bioenergetic function. These findings provide insights into synaptic mitochondrial susceptibility to damage. PMID:24708184
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Proteomes and Phosphoproteomes of Anther and Pollen: Availability and Progress.
Zhang, Zaibao; Hu, Menghui; Feng, Xiaobing; Gong, Andong; Cheng, Lin; Yuan, Hongyu
2017-10-01
In flowering plants, anther development plays crucial role in sexual reproduction. Within the anther, microspore mother cells meiosis produces microspores, which further develop into pollen grains that play decisive role in plant reproduction. Previous studies on anther biology mainly focused on single gene functions relying on genetic and molecular methods. Recently, anther development has been expanded from multiple OMICS approaches like transcriptomics, proteomics/phosphoproteomics, and metabolomics. The development of proteomics techniques allowing increased proteome coverage and quantitative measurements of proteins which can characterize proteomes and their modulation during normal development, biotic and abiotic stresses in anther development. In this review, we summarize the achievements of proteomics and phosphoproteomics with anther and pollen organs from model plant and crop species (i.e. Arabidopsis, rice, tobacco). The increased proteomic information facilitated translation of information from the models to crops and thus aid in agricultural improvement. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Tissue proteomics of the low-molecular weight proteome using an integrated cLC-ESI-QTOFMS approach.
Alvarez, MeiHwa Tanielle Bench; Shah, Dipti Jigar; Thulin, Craig D; Graves, Steven W
2013-05-01
Analysis of the protein/peptide composition of tissue has provided meaningful insights into tissue biology and even disease mechanisms. However, little has been published regarding top down methods to investigate lower molecular weight (MW) (500-5000 Da) species in tissue. Here, we evaluate a tissue proteomics approach involving tissue homogenization followed by depletion of large proteins and then cLC-MS (where c stands for capillary) analysis to interrogate the low MW/low abundance tissue proteome. In the development of this method, sheep heart, lung, liver, kidney, and spleen were surveyed to test our ability to observe tissue differences. After categorical tissue differences were demonstrated, a detailed study of this method's reproducibility was undertaken to determine whether or not it is suitable for analyzing more subtle differences in the abundance of small proteins and peptides. Our results suggest that this method should be useful in exploring the low MW proteome of tissues. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Urine Sample Preparation in 96-Well Filter Plates for Quantitative Clinical Proteomics
2015-01-01
Urine is an important, noninvasively collected body fluid source for the diagnosis and prognosis of human diseases. Liquid chromatography mass spectrometry (LC-MS) based shotgun proteomics has evolved as a sensitive and informative technique to discover candidate disease biomarkers from urine specimens. Filter-aided sample preparation (FASP) generates peptide samples from protein mixtures of cell lysate or body fluid origin. Here, we describe a FASP method adapted to 96-well filter plates, named 96FASP. Soluble urine concentrates containing ∼10 μg of total protein were processed by 96FASP and LC-MS resulting in 700–900 protein identifications at a 1% false discovery rate (FDR). The experimental repeatability, as assessed by label-free quantification and Pearson correlation analysis for shared proteins among replicates, was high (R ≥ 0.97). Application to urinary pellet lysates which is of particular interest in the context of urinary tract infection analysis was also demonstrated. On average, 1700 proteins (±398) were identified in five experiments. In a pilot study using 96FASP for analysis of eight soluble urine samples, we demonstrated that protein profiles of technical replicates invariably clustered; the protein profiles for distinct urine donors were very different from each other. Robust, highly parallel methods to generate peptide mixtures from urine and other body fluids are critical to increase cost-effectiveness in clinical proteomics projects. This 96FASP method has potential to become a gold standard for high-throughput quantitative clinical proteomics. PMID:24797144
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Picotti, Paola; Clement-Ziza, Mathieu; Lam, Henry; Campbell, David S.; Schmidt, Alexander; Deutsch, Eric W.; Röst, Hannes; Sun, Zhi; Rinner, Oliver; Reiter, Lukas; Shen, Qin; Michaelson, Jacob J.; Frei, Andreas; Alberti, Simon; Kusebauch, Ulrike; Wollscheid, Bernd; Moritz, Robert; Beyer, Andreas; Aebersold, Ruedi
2013-01-01
Complete reference maps or datasets, like the genomic map of an organism, are highly beneficial tools for biological and biomedical research. Attempts to generate such reference datasets for a proteome so far failed to reach complete proteome coverage, with saturation apparent at approximately two thirds of the proteomes tested, even for the most thoroughly characterized proteomes. Here, we used a strategy based on high-throughput peptide synthesis and mass spectrometry to generate a close to complete reference map (97% of the genome-predicted proteins) of the S. cerevisiae proteome. We generated two versions of this mass spectrometric map one supporting discovery- (shotgun) and the other hypothesis-driven (targeted) proteomic measurements. The two versions of the map, therefore, constitute a complete set of proteomic assays to support most studies performed with contemporary proteomic technologies. The reference libraries can be browsed via a web-based repository and associated navigation tools. To demonstrate the utility of the reference libraries we applied them to a protein quantitative trait locus (pQTL) analysis, which requires measurement of the same peptides over a large number of samples with high precision. Protein measurements over a set of 78 S. cerevisiae strains revealed a complex relationship between independent genetic loci, impacting on the levels of related proteins. Our results suggest that selective pressure favors the acquisition of sets of polymorphisms that maintain the stoichiometry of protein complexes and pathways. PMID:23334424
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Carpp, Lindsay N.; Rogers, Richard S.; Moritz, Robert L.; Aitchison, John D.
2014-01-01
Dengue virus is considered to be the most important mosquito-borne virus worldwide and poses formidable economic and health care burdens on many tropical and subtropical countries. Dengue infection induces drastic rearrangement of host endoplasmic reticulum membranes into complex membranous structures housing replication complexes; the contribution(s) of host proteins and pathways to this process is poorly understood but is likely to be mediated by protein-protein interactions. We have developed an approach for obtaining high confidence protein-protein interaction data by employing affinity tags and quantitative proteomics, in the context of viral infection, followed by robust statistical analysis. Using this approach, we identified high confidence interactors of NS5, the viral polymerase, and NS3, the helicase/protease. Quantitative proteomics allowed us to exclude a large number of presumably nonspecific interactors from our data sets and imparted a high level of confidence to our resulting data sets. We identified 53 host proteins reproducibly associated with NS5 and 41 with NS3, with 13 of these candidates present in both data sets. The host factors identified have diverse functions, including retrograde Golgi-to-endoplasmic reticulum transport, biosynthesis of long-chain fatty-acyl-coenzyme As, and in the unfolded protein response. We selected GBF1, a guanine nucleotide exchange factor responsible for ARF activation, from the NS5 data set for follow up and functional validation. We show that GBF1 plays a critical role early in dengue infection that is independent of its role in the maintenance of Golgi structure. Importantly, the approach described here can be applied to virtually any organism/system as a tool for better understanding its molecular interactions. PMID:24855065
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Jarnuczak, Andrew F; Lee, Dave C H; Lawless, Craig; Holman, Stephen W; Eyers, Claire E; Hubbard, Simon J
2016-09-02
Quantitative mass spectrometry-based proteomics of complex biological samples remains challenging in part due to the variability and charge competition arising during electrospray ionization (ESI) of peptides and the subsequent transfer and detection of ions. These issues preclude direct quantification from signal intensity alone in the absence of a standard. A deeper understanding of the governing principles of peptide ionization and exploitation of the inherent ionization and detection parameters of individual peptides is thus of great value. Here, using the yeast proteome as a model system, we establish the concept of peptide F-factor as a measure of detectability, closely related to ionization efficiency. F-factor is calculated by normalizing peptide precursor ion intensity by absolute abundance of the parent protein. We investigated F-factor characteristics in different shotgun proteomics experiments, including across multiple ESI-based LC-MS platforms. We show that F-factors mirror previously observed physicochemical predictors as peptide detectability but demonstrate a nonlinear relationship between hydrophobicity and peptide detectability. Similarly, we use F-factors to show how peptide ion coelution adversely affects detectability and ionization. We suggest that F-factors have great utility for understanding peptide detectability and gas-phase ion chemistry in complex peptide mixtures, selection of surrogate peptides in targeted MS studies, and for calibration of peptide ion signal in label-free workflows. Data are available via ProteomeXchange with identifier PXD003472.
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Boyanova, Desislava; Nilla, Santosh; Klau, Gunnar W.; Dandekar, Thomas; Müller, Tobias; Dittrich, Marcus
2014-01-01
The continuously evolving field of proteomics produces increasing amounts of data while improving the quality of protein identifications. Albeit quantitative measurements are becoming more popular, many proteomic studies are still based on non-quantitative methods for protein identification. These studies result in potentially large sets of identified proteins, where the biological interpretation of proteins can be challenging. Systems biology develops innovative network-based methods, which allow an integrated analysis of these data. Here we present a novel approach, which combines prior knowledge of protein-protein interactions (PPI) with proteomics data using functional similarity measurements of interacting proteins. This integrated network analysis exactly identifies network modules with a maximal consistent functional similarity reflecting biological processes of the investigated cells. We validated our approach on small (H9N2 virus-infected gastric cells) and large (blood constituents) proteomic data sets. Using this novel algorithm, we identified characteristic functional modules in virus-infected cells, comprising key signaling proteins (e.g. the stress-related kinase RAF1) and demonstrate that this method allows a module-based functional characterization of cell types. Analysis of a large proteome data set of blood constituents resulted in clear separation of blood cells according to their developmental origin. A detailed investigation of the T-cell proteome further illustrates how the algorithm partitions large networks into functional subnetworks each representing specific cellular functions. These results demonstrate that the integrated network approach not only allows a detailed analysis of proteome networks but also yields a functional decomposition of complex proteomic data sets and thereby provides deeper insights into the underlying cellular processes of the investigated system. PMID:24807868
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Burnum-Johnson, Kristin E.; Nie, Song; Casey, Cameron P.; Monroe, Matthew E.; Orton, Daniel J.; Ibrahim, Yehia M.; Gritsenko, Marina A.; Clauss, Therese R. W.; Shukla, Anil K.; Moore, Ronald J.; Purvine, Samuel O.; Shi, Tujin; Qian, Weijun; Liu, Tao; Baker, Erin S.; Smith, Richard D.
2016-01-01
Current proteomic approaches include both broad discovery measurements and quantitative targeted analyses. In many cases, discovery measurements are initially used to identify potentially important proteins (e.g. candidate biomarkers) and then targeted studies are employed to quantify a limited number of selected proteins. Both approaches, however, suffer from limitations. Discovery measurements aim to sample the whole proteome but have lower sensitivity, accuracy, and quantitation precision than targeted approaches, whereas targeted measurements are significantly more sensitive but only sample a limited portion of the proteome. Herein, we describe a new approach that performs both discovery and targeted monitoring (DTM) in a single analysis by combining liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled target peptides are spiked into tryptic digests and both the labeled and unlabeled peptides are detected using LC-IMS-MS instrumentation. Compared with the broad LC-MS discovery measurements, DTM yields greater peptide/protein coverage and detects lower abundance species. DTM also achieved detection limits similar to selected reaction monitoring (SRM) indicating its potential for combined high quality discovery and targeted analyses, which is a significant step toward the convergence of discovery and targeted approaches. PMID:27670688
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Comparative shotgun proteomics using spectral count data and quasi-likelihood modeling.
Li, Ming; Gray, William; Zhang, Haixia; Chung, Christine H; Billheimer, Dean; Yarbrough, Wendell G; Liebler, Daniel C; Shyr, Yu; Slebos, Robbert J C
2010-08-06
Shotgun proteomics provides the most powerful analytical platform for global inventory of complex proteomes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and allows a global analysis of protein changes. Nevertheless, sampling of complex proteomes by current shotgun proteomics platforms is incomplete, and this contributes to variability in assessment of peptide and protein inventories by spectral counting approaches. Thus, shotgun proteomics data pose challenges in comparing proteomes from different biological states. We developed an analysis strategy using quasi-likelihood Generalized Linear Modeling (GLM), included in a graphical interface software package (QuasiTel) that reads standard output from protein assemblies created by IDPicker, an HTML-based user interface to query shotgun proteomic data sets. This approach was compared to four other statistical analysis strategies: Student t test, Wilcoxon rank test, Fisher's Exact test, and Poisson-based GLM. We analyzed the performance of these tests to identify differences in protein levels based on spectral counts in a shotgun data set in which equimolar amounts of 48 human proteins were spiked at different levels into whole yeast lysates. Both GLM approaches and the Fisher Exact test performed adequately, each with their unique limitations. We subsequently compared the proteomes of normal tonsil epithelium and HNSCC using this approach and identified 86 proteins with differential spectral counts between normal tonsil epithelium and HNSCC. We selected 18 proteins from this comparison for verification of protein levels between the individual normal and tumor tissues using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS). This analysis confirmed the magnitude and direction of the protein expression differences in all 6 proteins for which reliable data could be obtained. Our analysis demonstrates that shotgun proteomic data sets from different tissue phenotypes are
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Comparative Shotgun Proteomics Using Spectral Count Data and Quasi-Likelihood Modeling
2010-01-01
Shotgun proteomics provides the most powerful analytical platform for global inventory of complex proteomes using liquid chromatography−tandem mass spectrometry (LC−MS/MS) and allows a global analysis of protein changes. Nevertheless, sampling of complex proteomes by current shotgun proteomics platforms is incomplete, and this contributes to variability in assessment of peptide and protein inventories by spectral counting approaches. Thus, shotgun proteomics data pose challenges in comparing proteomes from different biological states. We developed an analysis strategy using quasi-likelihood Generalized Linear Modeling (GLM), included in a graphical interface software package (QuasiTel) that reads standard output from protein assemblies created by IDPicker, an HTML-based user interface to query shotgun proteomic data sets. This approach was compared to four other statistical analysis strategies: Student t test, Wilcoxon rank test, Fisher’s Exact test, and Poisson-based GLM. We analyzed the performance of these tests to identify differences in protein levels based on spectral counts in a shotgun data set in which equimolar amounts of 48 human proteins were spiked at different levels into whole yeast lysates. Both GLM approaches and the Fisher Exact test performed adequately, each with their unique limitations. We subsequently compared the proteomes of normal tonsil epithelium and HNSCC using this approach and identified 86 proteins with differential spectral counts between normal tonsil epithelium and HNSCC. We selected 18 proteins from this comparison for verification of protein levels between the individual normal and tumor tissues using liquid chromatography−multiple reaction monitoring mass spectrometry (LC−MRM-MS). This analysis confirmed the magnitude and direction of the protein expression differences in all 6 proteins for which reliable data could be obtained. Our analysis demonstrates that shotgun proteomic data sets from different tissue
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2017-01-01
Myeloid cells play a central role in the context of viral eradication, yet precisely how these cells differentiate throughout the course of acute infections is poorly understood. In this study, we have developed a novel quantitative temporal in vivo proteomics (QTiPs) platform to capture proteomic signatures of temporally transitioning virus-driven myeloid cells directly in situ, thus taking into consideration host–virus interactions throughout the course of an infection. QTiPs, in combination with phenotypic, functional, and metabolic analyses, elucidated a pivotal role for inflammatory CD11b+, Ly6G–, Ly6Chigh-low cells in antiviral immune response and viral clearance. Most importantly, the time-resolved QTiPs data set showed the transition of CD11b+, Ly6G–, Ly6Chigh-low cells into M2-like macrophages, which displayed increased antigen-presentation capacities and bioenergetic demands late in infection. We elucidated the pivotal role of myeloid cells in virus clearance and show how these cells phenotypically, functionally, and metabolically undergo a timely transition from inflammatory to M2-like macrophages in vivo. With respect to the growing appreciation for in vivo examination of viral–host interactions and for the role of myeloid cells, this study elucidates the use of quantitative proteomics to reveal the role and response of distinct immune cell populations throughout the course of virus infection. PMID:28768414
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Chemical Proteomic Approaches Targeting Cancer Stem Cells: A Review of Current Literature.
Jung, Hye Jin
2017-01-01
Cancer stem cells (CSCs) have been proposed as central drivers of tumor initiation, progression, recurrence, and therapeutic resistance. Therefore, identifying stem-like cells within cancers and understanding their properties is crucial for the development of effective anticancer therapies. Recently, chemical proteomics has become a powerful tool to efficiently determine protein networks responsible for CSC pathophysiology and comprehensively elucidate molecular mechanisms of drug action against CSCs. This review provides an overview of major methodologies utilized in chemical proteomic approaches. In addition, recent successful chemical proteomic applications targeting CSCs are highlighted. Future direction of potential CSC research by integrating chemical genomic and proteomic data obtained from a single biological sample of CSCs are also suggested in this review. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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Ma, Yanlei; Zhang, Peng; Wang, Feng; Liu, Weijie; Yang, Jianjun; Qin, Huanlong
2012-04-01
The present study was designed to search for potential diagnostic biomarkers in the serum of colorectal cancer (CRC). CRC is the third most common cancer worldwide, and its prognosis is poor at early stages. A panel of novel biomarkers is urgently needed for early diagnosis of CRC. An integrated proteomics and metabolomics approach was performed to define oncofetal biomarkers in CRC by protein and metabolite profiling of serum samples from CRC patients, healthy control adults, and fetus. The differentially expressed proteins were identified by a 2-D DIGE (2-Dimensional Difference Gel Electrophoresis) coupled with a Finnigan LTQ-based proteomics approach. Meanwhile, the serum metabolome was analyzed using gas chromatography-mass spectrometry integrated with a commercial mass spectral library for peak identification. Of the 28 identified proteins and the 34 analyzed metabolites, only 5 protein spots and 6 metabolites were significantly increased or decreased in both CRC and fetal serum groups compared with the healthy adult group. Data from supervised predictive models allowed a separation of 93.5% of CRC patients from the healthy controls using the 6 metabolites. Finally, correlation analysis was applied to establish quantitative linkages between the 5 individual metabolite 3-hydroxybutyric acid, L-valine, L-threonine, 1-deoxyglucose, and glycine and the 5 individual proteins MACF1, APOH, A2M, IGL@, and VDB. Furthermore, 10 potential oncofetal biomarkers were characterized and their potential for CRC diagnosis was validated. The integrated approach we developed will promote the translation of biomarkers with clinical value into routine clinical practice.
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Yau, Yunki; Duo, Xizi; Zeng, Ming; Campbell, Beth; Shin, Sean; Luber, Raphael; Redmond, Diane; Leong, Rupert W. L.
2016-01-01
Breakdown of the protective gut barrier releases effector molecules and degradation products into the blood stream making serum and plasma ideal as a diagnostic medium. The enriched low mass proteome is unexplored as a source of differentiators for diagnosing and monitoring inflammatory bowel disease (IBD) activity, that is less invasive than colonoscopy. Differences in the enriched low mass plasma proteome (<25 kDa) were assessed by label-free quantitative mass-spectrometry. A panel of marker candidates were progressed to validation phase and “Tier-2” FDA-level validated quantitative assay. Proteins important in maintaining gut barrier function and homeostasis at the epithelial interface have been quantitated by multiple reaction monitoring in plasma and serum including both inflammatory; rheumatoid arthritis controls, and non-inflammatory healthy controls; ulcerative colitis (UC), and Crohn's disease (CD) patients. Detection by immunoblot confirmed presence at the protein level in plasma. Correlation analysis and receiver operator characteristics were used to report the sensitivity and specificity. Peptides differentiating controls from IBD originate from secreted phosphoprotein 24 (SPP24, p = 0.000086, 0.009); whereas those in remission and healthy can be differentiated in UC by SPP24 (p = 0.00023, 0.001), α-1-microglobulin (AMBP, p = 0.006) and CD by SPP24 (p = 0.019, 0.05). UC and CD can be differentiated by Guanylin (GUC2A, p = 0.001), and Secretogranin-1 (CHGB p = 0.035). Active and quiescent disease can also be differentiated in UC and CD by CHGB (p ≤ 0.023) SPP24 (p ≤ 0.023) and AMBP (UC p = 0.046). Five peptides discriminating IBD activity and severity had very little-to-no correlation to erythrocyte sedimentation rate, C-reactive protein, white cell or platelet counts. Three of these peptides were found to be binding partners to SPP24 protein alongside other known matrix proteins. These proteins have the potential to improve diagnosis and
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Schilling, Birgit; Gibson, Bradford W.; Hunter, Christie L.
2017-01-01
Data-independent acquisition is a powerful mass spectrometry technique that enables comprehensive MS and MS/MS analysis of all detectable species, providing an information rich data file that can be mined deeply. Here, we describe how to acquire high-quality SWATH® Acquisition data to be used for large quantitative proteomic studies. We specifically focus on using variable sized Q1 windows for acquisition of MS/MS data for generating higher specificity quantitative data. PMID:28188533
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Proteomic approaches to understanding the role of the cytoskeleton in host-defense mechanisms
Radulovic, Marko; Godovac-Zimmermann, Jasminka
2014-01-01
The cytoskeleton is a cellular scaffolding system whose functions include maintenance of cellular shape, enabling cellular migration, division, intracellular transport, signaling and membrane organization. In addition, in immune cells, the cytoskeleton is essential for phagocytosis. Following the advances in proteomics technology over the past two decades, cytoskeleton proteome analysis in resting and activated immune cells has emerged as a possible powerful approach to expand our understanding of cytoskeletal composition and function. However, so far there have only been a handful of studies of the cytoskeleton proteome in immune cells. This article considers promising proteomics strategies that could augment our understanding of the role of the cytoskeleton in host-defense mechanisms. PMID:21329431
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iTRAQ Quantitative Proteomic Comparison of Metastatic and Non-Metastatic Uveal Melanoma Tumors
Crabb, John W.; Hu, Bo; Crabb, John S.; Triozzi, Pierre; Saunthararajah, Yogen; Singh, Arun D.
2015-01-01
Background Uveal melanoma is the most common malignancy of the adult eye. The overall mortality rate is high because this aggressive cancer often metastasizes before ophthalmic diagnosis. Quantitative proteomic analysis of primary metastasizing and non-metastasizing tumors was pursued for insights into mechanisms and biomarkers of uveal melanoma metastasis. Methods Eight metastatic and 7 non-metastatic human primary uveal melanoma tumors were analyzed by LC MS/MS iTRAQ technology with Bruch’s membrane/choroid complex from normal postmortem eyes as control tissue. Tryptic peptides from tumor and control proteins were labeled with iTRAQ tags, fractionated by cation exchange chromatography, and analyzed by LC MS/MS. Protein identification utilized the Mascot search engine and the human Uni-Prot/Swiss-Protein database with false discovery ≤ 1%; protein quantitation utilized the Mascot weighted average method. Proteins designated differentially expressed exhibited quantitative differences (p ≤ 0.05, t-test) in a training set of five metastatic and five non-metastatic tumors. Logistic regression models developed from the training set were used to classify the metastatic status of five independent tumors. Results Of 1644 proteins identified and quantified in 5 metastatic and 5 non-metastatic tumors, 12 proteins were found uniquely in ≥ 3 metastatic tumors, 28 were found significantly elevated and 30 significantly decreased only in metastatic tumors, and 31 were designated differentially expressed between metastatic and non-metastatic tumors. Logistic regression modeling of differentially expressed collagen alpha-3(VI) and heat shock protein beta-1 allowed correct prediction of metastasis status for each of five independent tumor specimens. Conclusions The present data provide new clues to molecular differences in metastatic and non-metastatic uveal melanoma tumors. While sample size is limited and validation required, the results support collagen alpha-3(VI) and
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Identification of Tengfu Jiangya Tablet Target Biomarkers with Quantitative Proteomic Technique
Xu, Jingwen; Zhang, Shijun; Jiang, Haiqiang; Wang, Nan; Lin, Haiqing
2017-01-01
Tengfu Jiangya Tablet (TJT) is a well accepted antihypertension drug in China and its major active components were Uncaria total alkaloids and Semen Raphani soluble alkaloid. To further explore treatment effects mechanism of TJT on essential hypertension, a serum proteomic study was performed. Potential biomarkers were quantified in serum of hypertension individuals before and after taking TJT with isobaric tags for relative and absolute quantitation (iTRAQ) coupled two-dimensional liquid chromatography followed electrospray ionization-tandem mass spectrometry (2D LC-MS/MS) proteomics technique. Among 391 identified proteins with high confidence, 70 proteins were differentially expressed (fold variation criteria, >1.2 or <0.83) between two groups (39 upregulated and 31 downregulated). Combining with Gene Ontology annotation, KEGG pathway analysis, and literature retrieval, 5 proteins were chosen as key target biomarkers during TJT therapeutic process. And the alteration profiles of these 5 proteins were verified by ELISA and Western Blot. Proteins Kininogen 1 and Keratin 1 are members of Kallikrein system, while Myeloperoxidase, Serum Amyloid protein A, and Retinol binding protein 4 had been reported closely related to vascular endothelial injury. Our study discovered 5 target biomarkers of the compound Chinese medicine TJT. Secondly, this research initially revealed the antihypertension therapeutic mechanism of this drug from a brand-new aspect. PMID:28408942
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Kuzniar, Arnold; Laffeber, Charlie; Eppink, Berina; Bezstarosti, Karel; Dekkers, Dick; Woelders, Henri; Zwamborn, A Peter M; Demmers, Jeroen; Lebbink, Joyce H G; Kanaar, Roland
2017-01-01
The potential effects of non-ionizing electromagnetic fields (EMFs), such as those emitted by power-lines (in extremely low frequency range), mobile cellular systems and wireless networking devices (in radio frequency range) on human health have been intensively researched and debated. However, how exposure to these EMFs may lead to biological changes underlying possible health effects is still unclear. To reveal EMF-induced molecular changes, unbiased experiments (without a priori focusing on specific biological processes) with sensitive readouts are required. We present the first proteome-wide semi-quantitative mass spectrometry analysis of human fibroblasts, osteosarcomas and mouse embryonic stem cells exposed to three types of non-ionizing EMFs (ELF 50 Hz, UMTS 2.1 GHz and WiFi 5.8 GHz). We performed controlled in vitro EMF exposures of metabolically labeled mammalian cells followed by reliable statistical analyses of differential protein- and pathway-level regulations using an array of established bioinformatics methods. Our results indicate that less than 1% of the quantitated human or mouse proteome responds to the EMFs by small changes in protein abundance. Further network-based analysis of the differentially regulated proteins did not detect significantly perturbed cellular processes or pathways in human and mouse cells in response to ELF, UMTS or WiFi exposure. In conclusion, our extensive bioinformatics analyses of semi-quantitative mass spectrometry data do not support the notion that the short-time exposures to non-ionizing EMFs have a consistent biologically significant bearing on mammalian cells in culture.
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Laffeber, Charlie; Eppink, Berina; Bezstarosti, Karel; Dekkers, Dick; Woelders, Henri; Zwamborn, A. Peter M.; Demmers, Jeroen; Lebbink, Joyce H. G.; Kanaar, Roland
2017-01-01
The potential effects of non-ionizing electromagnetic fields (EMFs), such as those emitted by power-lines (in extremely low frequency range), mobile cellular systems and wireless networking devices (in radio frequency range) on human health have been intensively researched and debated. However, how exposure to these EMFs may lead to biological changes underlying possible health effects is still unclear. To reveal EMF-induced molecular changes, unbiased experiments (without a priori focusing on specific biological processes) with sensitive readouts are required. We present the first proteome-wide semi-quantitative mass spectrometry analysis of human fibroblasts, osteosarcomas and mouse embryonic stem cells exposed to three types of non-ionizing EMFs (ELF 50 Hz, UMTS 2.1 GHz and WiFi 5.8 GHz). We performed controlled in vitro EMF exposures of metabolically labeled mammalian cells followed by reliable statistical analyses of differential protein- and pathway-level regulations using an array of established bioinformatics methods. Our results indicate that less than 1% of the quantitated human or mouse proteome responds to the EMFs by small changes in protein abundance. Further network-based analysis of the differentially regulated proteins did not detect significantly perturbed cellular processes or pathways in human and mouse cells in response to ELF, UMTS or WiFi exposure. In conclusion, our extensive bioinformatics analyses of semi-quantitative mass spectrometry data do not support the notion that the short-time exposures to non-ionizing EMFs have a consistent biologically significant bearing on mammalian cells in culture. PMID:28234898
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Maillard Proteomics: Opening New Pages
Soboleva, Alena; Schmidt, Rico; Vikhnina, Maria; Grishina, Tatiana; Frolov, Andrej
2017-01-01
Protein glycation is a ubiquitous non-enzymatic post-translational modification, formed by reaction of protein amino and guanidino groups with carbonyl compounds, presumably reducing sugars and α-dicarbonyls. Resulting advanced glycation end products (AGEs) represent a highly heterogeneous group of compounds, deleterious in mammals due to their pro-inflammatory effect, and impact in pathogenesis of diabetes mellitus, Alzheimer’s disease and ageing. The body of information on the mechanisms and pathways of AGE formation, acquired during the last decades, clearly indicates a certain site-specificity of glycation. It makes characterization of individual glycation sites a critical pre-requisite for understanding in vivo mechanisms of AGE formation and developing adequate nutritional and therapeutic approaches to reduce it in humans. In this context, proteomics is the methodology of choice to address site-specific molecular changes related to protein glycation. Therefore, here we summarize the methods of Maillard proteomics, specifically focusing on the techniques providing comprehensive structural and quantitative characterization of glycated proteome. Further, we address the novel break-through areas, recently established in the field of Maillard research, i.e., in vitro models based on synthetic peptides, site-based diagnostics of metabolism-related diseases (e.g., diabetes mellitus), proteomics of anti-glycative defense, and dynamics of plant glycated proteome during ageing and response to environmental stress. PMID:29231845
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Proteomic approaches in brain research and neuropharmacology.
Vercauteren, Freya G G; Bergeron, John J M; Vandesande, Frans; Arckens, Lut; Quirion, Rémi
2004-10-01
Numerous applications of genomic technologies have enabled the assembly of unprecedented inventories of genes, expressed in cells under specific physiological and pathophysiological conditions. Complementing the valuable information generated through functional genomics with the integrative knowledge of protein expression and function should enable the development of more efficient diagnostic tools and therapeutic agents. Proteomic analyses are particularly suitable to elucidate posttranslational modifications, expression levels and protein-protein interactions of thousands of proteins at a time. In this review, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) investigations of brain tissues in neurodegenerative diseases such as Alzheimer's disease, Down syndrome and schizophrenia, and the construction of 2D-PAGE proteome maps of the brain are discussed. The role of the Human Proteome Organization (HUPO) as an international coordinating organization for proteomic efforts, as well as challenges for proteomic technologies and data analysis are also addressed. It is expected that the use of proteomic strategies will have significant impact in neuropharmacology over the coming decade.
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Advances in Proteomics Data Analysis and Display Using an Accurate Mass and Time Tag Approach
Zimmer, Jennifer S.D.; Monroe, Matthew E.; Qian, Wei-Jun; Smith, Richard D.
2007-01-01
Proteomics has recently demonstrated utility in understanding cellular processes on the molecular level as a component of systems biology approaches and for identifying potential biomarkers of various disease states. The large amount of data generated by utilizing high efficiency (e.g., chromatographic) separations coupled to high mass accuracy mass spectrometry for high-throughput proteomics analyses presents challenges related to data processing, analysis, and display. This review focuses on recent advances in nanoLC-FTICR-MS-based proteomics approaches and the accompanying data processing tools that have been developed to display and interpret the large volumes of data being produced. PMID:16429408
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Ye, Dongping; Liang, Weiguo; Dai, Libing; Zhou, Longqiang; Yao, Yicun; Zhong, Xin; Chen, Honghui; Xu, Jiake
2015-05-01
Degeneration of the intervertebral disc (IVD) is a major chronic medical condition associated with back pain. To better understand the pathogenesis of IVD degeneration, we performed comparative and quantitative proteomic analyses of normal and degenerated human annulus fibrosus (AF) cells and identified proteins that are differentially expressed between them. Annulus fibrosus cells were isolated and cultured from patients with lumbar disc herniation (the experimental group, degenerated AF cells) and scoliosis patients who underwent orthopaedic surgery (the control group, normal AF cells). Comparative proteomic analyses of normal and degenerated cultured AF cells were carried out using 2-D electrophoresis, mass spectrometric analyses, and database searching. Quantitative analyses of silver-stained 2-D electrophoresis gels of normal and degenerated cultured AF cells identified 10 protein spots that showed the most altered differential expression levels between the two groups. Among these, three proteins were decreased, including heat shock cognate 71-kDa protein, glucose-6-phosphate 1-dehydrogenase, and protocadherin-23, whereas seven proteins were increased, including guanine nucleotide-binding protein G(i) subunit α-2, superoxide dismutase, transmembrane protein 51, adenosine receptor A3, 26S protease regulatory subunit 8, lipid phosphate phosphatase-related protein, and fatty acyl-crotonic acid reductase 1. These differentially expressed proteins might be involved in the pathophysiological process of IVD degeneration and have potential values as biomarkers of the degeneration of IVD. © 2015 Wiley Publishing Asia Pty Ltd.
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Targeted proteomics identifies liquid-biopsy signatures for extracapsular prostate cancer
Kim, Yunee; Jeon, Jouhyun; Mejia, Salvador; Yao, Cindy Q; Ignatchenko, Vladimir; Nyalwidhe, Julius O; Gramolini, Anthony O; Lance, Raymond S; Troyer, Dean A; Drake, Richard R; Boutros, Paul C; Semmes, O. John; Kislinger, Thomas
2016-01-01
Biomarkers are rapidly gaining importance in personalized medicine. Although numerous molecular signatures have been developed over the past decade, there is a lack of overlap and many biomarkers fail to validate in independent patient cohorts and hence are not useful for clinical application. For these reasons, identification of novel and robust biomarkers remains a formidable challenge. We combine targeted proteomics with computational biology to discover robust proteomic signatures for prostate cancer. Quantitative proteomics conducted in expressed prostatic secretions from men with extraprostatic and organ-confined prostate cancers identified 133 differentially expressed proteins. Using synthetic peptides, we evaluate them by targeted proteomics in a 74-patient cohort of expressed prostatic secretions in urine. We quantify a panel of 34 candidates in an independent 207-patient cohort. We apply machine-learning approaches to develop clinical predictive models for prostate cancer diagnosis and prognosis. Our results demonstrate that computationally guided proteomics can discover highly accurate non-invasive biomarkers. PMID:27350604
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Biomarker Discovery and Mechanistic Studies of Prostate Cancer Using Targeted Proteomic Approaches
2010-07-01
1-0431 TITLE: Biomarker Discovery and Mechanistic Studies of Prostate Cancer Using Targeted Proteomic Approaches PRINCIPAL INVESTIGATOR...June 2010 4. TITLE AND SUBTITLE Biomarker Discovery and Mechanistic Studies of Prostate Cancer Using Targeted Proteomic 5a. CONTRACT NUMBER...1-0430; W81XWH-08-1-0431; Grant sponsor: NIH/NCRR COBRE Grant; Grant number: 1P20RR020171; Grant sponsor: NIH/NIDDK Grant; Grant number: R01DK053525
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Recent advances in mass spectrometry-based proteomics of gastric cancer.
Kang, Changwon; Lee, Yejin; Lee, J Eugene
2016-10-07
The last decade has witnessed remarkable technological advances in mass spectrometry-based proteomics. The development of proteomics techniques has enabled the reliable analysis of complex proteomes, leading to the identification and quantification of thousands of proteins in gastric cancer cells, tissues, and sera. This quantitative information has been used to profile the anomalies in gastric cancer and provide insights into the pathogenic mechanism of the disease. In this review, we mainly focus on the advances in mass spectrometry and quantitative proteomics that were achieved in the last five years and how these up-and-coming technologies are employed to track biochemical changes in gastric cancer cells. We conclude by presenting a perspective on quantitative proteomics and its future applications in the clinic and translational gastric cancer research.
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Tiberti, Natalia; Sanchez, Jean-Charles
2015-09-01
The quantitative proteomics data here reported are part of a research article entitled "Increased acute immune response during the meningo-encephalitic stage of Trypanosoma brucei rhodesiense sleeping sickness compared to Trypanosoma brucei gambiense", published by Tiberti et al., 2015. Transl. Proteomics 6, 1-9. Sleeping sickness (human African trypanosomiasis - HAT) is a deadly neglected tropical disease affecting mainly rural communities in sub-Saharan Africa. This parasitic disease is caused by the Trypanosoma brucei (T. b.) parasite, which is transmitted to the human host through the bite of the tse-tse fly. Two parasite sub-species, T. b. rhodesiense and T. b. gambiense, are responsible for two clinically different and geographically separated forms of sleeping sickness. The objective of the present study was to characterise and compare the cerebrospinal fluid (CSF) proteome of stage 2 (meningo-encephalitic stage) HAT patients suffering from T. b. gambiense or T. b. rhodesiense disease using high-throughput quantitative proteomics and the Tandem Mass Tag (TMT(®)) isobaric labelling. In order to evaluate the CSF proteome in the context of HAT pathophysiology, the protein dataset was then submitted to gene ontology and pathway analysis. Two significantly differentially expressed proteins (C-reactive protein and orosomucoid 1) were further verified on a larger population of patients (n=185) by ELISA, confirming the mass spectrometry results. By showing a predominant involvement of the acute immune response in rhodesiense HAT, the proteomics results obtained in this work will contribute to further understand the mechanisms of pathology occurring in HAT and to propose new biomarkers of potential clinical utility. The mass spectrometry raw data are available in the Pride Archive via ProteomeXchange through the identifier PXD001082.
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Moore, Henna M; Bai, Baoyan; Boisvert, François-Michel; Latonen, Leena; Rantanen, Ville; Simpson, Jeremy C; Pepperkok, Rainer; Lamond, Angus I; Laiho, Marikki
2011-10-01
The nucleolus is a nuclear organelle that coordinates rRNA transcription and ribosome subunit biogenesis. Recent proteomic analyses have shown that the nucleolus contains proteins involved in cell cycle control, DNA processing and DNA damage response and repair, in addition to the many proteins connected with ribosome subunit production. Here we study the dynamics of nucleolar protein responses in cells exposed to stress and DNA damage caused by ionizing and ultraviolet (UV) radiation in diploid human fibroblasts. We show using a combination of imaging and quantitative proteomics methods that nucleolar substructure and the nucleolar proteome undergo selective reorganization in response to UV damage. The proteomic responses to UV include alterations of functional protein complexes such as the SSU processome and exosome, and paraspeckle proteins, involving both decreases and increases in steady state protein ratios, respectively. Several nonhomologous end-joining proteins (NHEJ), such as Ku70/80, display similar fast responses to UV. In contrast, nucleolar proteomic responses to IR are both temporally and spatially distinct from those caused by UV, and more limited in terms of magnitude. With the exception of the NHEJ and paraspeckle proteins, where IR induces rapid and transient changes within 15 min of the damage, IR does not alter the ratios of most other functional nucleolar protein complexes. The rapid transient decrease of NHEJ proteins in the nucleolus indicates that it may reflect a response to DNA damage. Our results underline that the nucleolus is a specific stress response organelle that responds to different damage and stress agents in a unique, damage-specific manner.
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High-Resolution Enabled 12-Plex DiLeu Isobaric Tags for Quantitative Proteomics
2015-01-01
Multiplex isobaric tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ)) are a valuable tool for high-throughput mass spectrometry based quantitative proteomics. We have developed our own multiplex isobaric tags, DiLeu, that feature quantitative performance on par with commercial offerings but can be readily synthesized in-house as a cost-effective alternative. In this work, we achieve a 3-fold increase in the multiplexing capacity of the DiLeu reagent without increasing structural complexity by exploiting mass defects that arise from selective incorporation of 13C, 15N, and 2H stable isotopes in the reporter group. The inclusion of eight new reporter isotopologues that differ in mass from the existing four reporters by intervals of 6 mDa yields a 12-plex isobaric set that preserves the synthetic simplicity and quantitative performance of the original implementation. We show that the new reporter variants can be baseline-resolved in high-resolution higher-energy C-trap dissociation (HCD) spectra, and we demonstrate accurate 12-plex quantitation of a DiLeu-labeled Saccharomyces cerevisiae lysate digest via high-resolution nano liquid chromatography–tandem mass spectrometry (nanoLC–MS2) analysis on an Orbitrap Elite mass spectrometer. PMID:25405479
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Burnum-Johnson, Kristin E.; Nie, Song; Casey, Cameron P.
Current proteomics approaches are comprised of both broad discovery measurements as well as more quantitative targeted measurements. These two different measurement types are used to initially identify potentially important proteins (e.g., candidate biomarkers) and then enable improved quantification for a limited number of selected proteins. However, both approaches suffer from limitations, particularly the lower sensitivity, accuracy, and quantitation precision for discovery approaches compared to targeted approaches, and the limited proteome coverage provided by targeted approaches. Herein, we describe a new proteomics approach that allows both discovery and targeted monitoring (DTM) in a single analysis using liquid chromatography, ion mobility spectrometrymore » and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled peptides for target ions are spiked into tryptic digests and both the labeled and unlabeled peptides are broadly detected using LC-IMS-MS instrumentation, allowing the benefits of discovery and targeted approaches. To understand the possible improvement of the DTM approach, it was compared to LC-MS broad measurements using an accurate mass and time tag database and selected reaction monitoring (SRM) targeted measurements. The DTM results yielded greater peptide/protein coverage and a significant improvement in the detection of lower abundance species compared to LC-MS discovery measurements. DTM was also observed to have similar detection limits as SRM for the targeted measurements indicating its potential for combining the discovery and targeted approaches.« less
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Monitoring Peptidase Activities in Complex Proteomes by MALDI-TOF Mass Spectrometry
Villanueva, Josep; Nazarian, Arpi; Lawlor, Kevin; Tempst, Paul
2009-01-01
Measuring enzymatic activities in biological fluids is a form of activity-based proteomics and may be utilized as a means of developing disease biomarkers. Activity-based assays allow amplification of output signals, thus potentially visualizing low-abundant enzymes on a virtually transparent whole-proteome background. The protocol presented here describes a semi-quantitative in vitro assay of proteolytic activities in complex proteomes by monitoring breakdown of designer peptide-substrates using robotic extraction and a MALDI-TOF mass spectrometric read-out. Relative quantitation of the peptide metabolites is done by comparison with spiked internal standards, followed by statistical analysis of the resulting mini-peptidome. Partial automation provides reproducibility and throughput essential for comparing large sample sets. The approach may be employed for diagnostic or predictive purposes and enables profiling of 96 samples in 30 hours. It could be tailored to many diagnostic and pharmaco-dynamic purposes, as a read-out of catalytic and metabolic activities in body fluids or tissues. PMID:19617888
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Du, Chao; van Wezel, Gilles P
2018-04-30
Natural products (NPs) are a major source of compounds for medical, agricultural, and biotechnological industries. Many of these compounds are of microbial origin, and, in particular, from Actinobacteria or filamentous fungi. To successfully identify novel compounds that correlate to a bioactivity of interest, or discover new enzymes with desired functions, systematic multiomics approaches have been developed over the years. Bioinformatics tools harness the rapidly expanding wealth of genome sequence information, revealing previously unsuspected biosynthetic diversity. Varying growth conditions or application of elicitors are applied to activate cryptic biosynthetic gene clusters, and metabolomics provide detailed insights into the NPs they specify. Combining these technologies with proteomics-based approaches to profile the biosynthetic enzymes provides scientists with insights into the full biosynthetic potential of microorganisms. The proteomics approaches include enrichment strategies such as employing activity-based probes designed by chemical biology, as well as unbiased (quantitative) proteomics methods. In this review, the opportunities and challenges in microbial NP research are discussed, and, in particular, the application of proteomics to link biosynthetic enzymes to the molecules they produce, and vice versa. © 2018 The Authors. Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Nakayasu, Ernesto S.; Sydor, Michael A.; Brown, Roslyn N.; ...
2015-07-06
Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification and global quantitative proteomic analysis. As model system to identify substrates, we used a virulence-related deubiquitinase secreted by Salmonella enterica serovar Typhimurium into the host cells, SseL. By using this approach two SseL substrates were identified in RAW 264.7 murine macrophage-like cell line, S100A6 and het-erogeneous nuclear ribonuclear protein K, inmore » addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. Finally, this method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Nakayasu, Ernesto S.; Sydor, Michael A.; Brown, Roslyn N.
Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification and global quantitative proteomic analysis. As model system to identify substrates, we used a virulence-related deubiquitinase secreted by Salmonella enterica serovar Typhimurium into the host cells, SseL. By using this approach two SseL substrates were identified in RAW 264.7 murine macrophage-like cell line, S100A6 and het-erogeneous nuclear ribonuclear protein K, inmore » addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. Finally, this method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.« less
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Zhai, Linhui; Chang, Cheng; Li, Ning; Duong, Duc M; Chen, Hao; Deng, Zixin; Yang, Jian; Hong, Xuechuan; Zhu, Yunping; Xu, Ping
2013-08-01
Reversed phase microcolumns have been widely used for peptide pretreatment to desalt and remove interferences before tandem LC-MS in proteomics studies. However, few studies have characterized the effects of experimental parameters as well as column characteristics on the composition of identified peptides. In this study, several parameters including the concentration of ACN in washing buffer, the microcolumn's purification effect, the peptide recovery rate, and the dynamic-binding capacity were characterized in detail, based upon stable isotope labeling by amino acids in a cell culture quantitative approach. The results showed that peptide losses can be reduced with low ACN concentration in washing buffers resulting in a recovery rate of approximately 82%. Furthermore, the effects of ACN concentration and loading amount on the properties of identified peptides were also evaluated. We found that the dynamic-binding capacity of the column was approximately 26 μg. With increased loading amounts, more hydrophilic peptides were replaced by hydrophobic peptides. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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A proteomics performance standard to support measurement quality in proteomics.
Beasley-Green, Ashley; Bunk, David; Rudnick, Paul; Kilpatrick, Lisa; Phinney, Karen
2012-04-01
The emergence of MS-based proteomic platforms as a prominent technology utilized in biochemical and biomedical research has increased the need for high-quality MS measurements. To address this need, National Institute of Standards and Technology (NIST) reference material (RM) 8323 yeast protein extract is introduced as a proteomics quality control material for benchmarking the preanalytical and analytical performance of proteomics-based experimental workflows. RM 8323 yeast protein extract is based upon the well-characterized eukaryote Saccharomyces cerevisiae and can be utilized in the design and optimization of proteomics-based methodologies from sample preparation to data analysis. To demonstrate its utility as a proteomics quality control material, we coupled LC-MS/MS measurements of RM 8323 with the NIST MS Quality Control (MSQC) performance metrics to quantitatively assess the LC-MS/MS instrumentation parameters that influence measurement accuracy, repeatability, and reproducibility. Due to the complexity of the yeast proteome, we also demonstrate how NIST RM 8323, along with the NIST MSQC performance metrics, can be used in the evaluation and optimization of proteomics-based sample preparation methods. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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NASA Astrophysics Data System (ADS)
Zhao, Minzhi; Li, Haiyun; Liu, Xiaochen; Wei, Jie; Ji, Jianguo; Yang, Shu; Hu, Zhiyuan; Wei, Shicheng
2016-03-01
Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn’t increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca2+ concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery.
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Zhao, Minzhi; Li, Haiyun; Liu, Xiaochen; Wei, Jie; Ji, Jianguo; Yang, Shu; Hu, Zhiyuan; Wei, Shicheng
2016-03-09
Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn't increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca(2+) concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery.
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Martinez-Pinna, Roxana; Gonzalez de Peredo, Anne; Monsarrat, Bernard; Burlet-Schiltz, Odile; Martin-Ventura, Jose Luis
2014-08-01
To find potential biomarkers of abdominal aortic aneurysms (AAA), we performed a differential proteomic study based on human plasma-derived microvesicles. Exosomes and microparticles isolated from plasma of AAA patients and control subjects (n = 10 each group) were analyzed by a label-free quantitative MS-based strategy. Homemade and publicly available software packages have been used for MS data analysis. The application of two kinds of bioinformatic tools allowed us to find differential protein profiles from AAA patients. Some of these proteins found by the two analysis methods belong to main pathological mechanisms of AAA such as oxidative stress, immune-inflammation, and thrombosis. Data analysis from label-free MS-based experiments requires the use of sophisticated bioinformatic approaches to perform quantitative studies from complex protein mixtures. The application of two of these bioinformatic tools provided us a preliminary list of differential proteins found in plasma-derived microvesicles not previously associated to AAA, which could help us to understand the pathological mechanisms related to this disease. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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A Scalable Approach for Protein False Discovery Rate Estimation in Large Proteomic Data Sets
Savitski, Mikhail M.; Wilhelm, Mathias; Hahne, Hannes; Kuster, Bernhard; Bantscheff, Marcus
2015-01-01
Calculating the number of confidently identified proteins and estimating false discovery rate (FDR) is a challenge when analyzing very large proteomic data sets such as entire human proteomes. Biological and technical heterogeneity in proteomic experiments further add to the challenge and there are strong differences in opinion regarding the conceptual validity of a protein FDR and no consensus regarding the methodology for protein FDR determination. There are also limitations inherent to the widely used classic target–decoy strategy that particularly show when analyzing very large data sets and that lead to a strong over-representation of decoy identifications. In this study, we investigated the merits of the classic, as well as a novel target–decoy-based protein FDR estimation approach, taking advantage of a heterogeneous data collection comprised of ∼19,000 LC-MS/MS runs deposited in ProteomicsDB (https://www.proteomicsdb.org). The “picked” protein FDR approach treats target and decoy sequences of the same protein as a pair rather than as individual entities and chooses either the target or the decoy sequence depending on which receives the highest score. We investigated the performance of this approach in combination with q-value based peptide scoring to normalize sample-, instrument-, and search engine-specific differences. The “picked” target–decoy strategy performed best when protein scoring was based on the best peptide q-value for each protein yielding a stable number of true positive protein identifications over a wide range of q-value thresholds. We show that this simple and unbiased strategy eliminates a conceptual issue in the commonly used “classic” protein FDR approach that causes overprediction of false-positive protein identification in large data sets. The approach scales from small to very large data sets without losing performance, consistently increases the number of true-positive protein identifications and is readily
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Burnum-Johnson, Kristin E; Nie, Song; Casey, Cameron P; Monroe, Matthew E; Orton, Daniel J; Ibrahim, Yehia M; Gritsenko, Marina A; Clauss, Therese R W; Shukla, Anil K; Moore, Ronald J; Purvine, Samuel O; Shi, Tujin; Qian, Weijun; Liu, Tao; Baker, Erin S; Smith, Richard D
2016-12-01
Current proteomic approaches include both broad discovery measurements and quantitative targeted analyses. In many cases, discovery measurements are initially used to identify potentially important proteins (e.g. candidate biomarkers) and then targeted studies are employed to quantify a limited number of selected proteins. Both approaches, however, suffer from limitations. Discovery measurements aim to sample the whole proteome but have lower sensitivity, accuracy, and quantitation precision than targeted approaches, whereas targeted measurements are significantly more sensitive but only sample a limited portion of the proteome. Herein, we describe a new approach that performs both discovery and targeted monitoring (DTM) in a single analysis by combining liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled target peptides are spiked into tryptic digests and both the labeled and unlabeled peptides are detected using LC-IMS-MS instrumentation. Compared with the broad LC-MS discovery measurements, DTM yields greater peptide/protein coverage and detects lower abundance species. DTM also achieved detection limits similar to selected reaction monitoring (SRM) indicating its potential for combined high quality discovery and targeted analyses, which is a significant step toward the convergence of discovery and targeted approaches. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Soulet, Fabienne; Kilarski, Witold W.; Roux-Dalvai, Florence; Herbert, John M. J.; Sacewicz, Izabela; Mouton-Barbosa, Emmanuelle; Bicknell, Roy; Lalor, Patricia; Monsarrat, Bernard; Bikfalvi, Andreas
2013-01-01
In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in vivo, we developed a biotinylation technique for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in vivo biotinylation, in combination with robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that might constitute the starting point for further developments. PMID:23674615
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Xing, Xiaohua; Huang, Yao; Wang, Sen; Chi, Minhui; Zeng, Yongyi; Chen, Lihong; Li, Ling; Zeng, Jinhua; Lin, Minjie; Han, Xiao; Liu, Xiaolong; Liu, Jingfeng
2015-10-14
In clinical practices, the therapeutic outcomes and prognosis of hepatocellular carcinoma (HCC) patients with different tumor numbers after surgery are very different; however, the underlying mechanisms of the tumorigenesis and development of HCC with different tumor numbers are still not well understood. Here, we systematically compared the overall proteome profiles between the primary HCC with single and multiple lesions using iTRAQ-based quantitative proteomics approach. We identified that 107 and 330 proteins were dysregulated in HCC tissue with multiple lesions (MC group) and HCC tissue with a single lesion (SC group), compared with their non-cancerous tissue (MN and SN groups) respectively. The dysregulated proteins in MC group are concentrated in UBC signaling pathway and NFκB signaling pathway, but the dysregulated proteins in SC group are more concentrated in ERK signaling pathway and the NFκB signaling pathway. These information revealed that there might be different molecular mechanisms of the tumorigenesis and development of the HCC with single and multiple lesions. Furthermore, HSD17B13 were only down-regulated in MC group while HK2 were only up-regulated in SC group among these dysregulated proteins. Therefore, the protein HSD17B13 and HK2 might be potential biomarkers for the primary HCC with single and multiple lesions. Copyright © 2015 Elsevier B.V. All rights reserved.
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Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics
Bennett, Eric J.; Rush, John; Gygi, Steven P.; Harper, J. Wade
2010-01-01
Dynamic reorganization of signaling systems frequently accompany pathway perturbations, yet quantitative studies of network remodeling by pathway stimuli are lacking. Here, we report the development of a quantitative proteomics platform centered on multiplex Absolute Quantification (AQUA) technology to elucidate the architecture of the cullin-RING ubiquitin ligase (CRL) network and to evaluate current models of dynamic CRL remodeling. Current models suggest that CRL complexes are controlled by cycles of CRL deneddylation and CAND1 binding. Contrary to expectations, acute CRL inhibition with MLN4924, an inhibitor of the NEDD8-activating enzyme, does not result in a global reorganization of the CRL network. Examination of CRL complex stoichiometry reveals that, independent of cullin neddylation, a large fraction of cullins are assembled with adaptor modules while only a small fraction are associated with CAND1. These studies suggest an alternative model of CRL dynamicity where the abundance of adaptor modules, rather than cycles of neddylation and CAND1 binding, drives CRL network organization. PMID:21145461
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Burillo, Elena; Jorge, Inmaculada; Martínez-López, Diego; Camafeita, Emilio; Blanco-Colio, Luis Miguel; Trevisan-Herraz, Marco; Ezkurdia, Iakes; Egido, Jesús; Michel, Jean-Baptiste; Meilhac, Olivier; Vázquez, Jesús; Martin-Ventura, Jose Luis
2016-01-01
High-density lipoproteins (HDLs) are complex protein and lipid assemblies whose composition is known to change in diverse pathological situations. Analysis of the HDL proteome can thus provide insight into the main mechanisms underlying abdominal aortic aneurysm (AAA) and potentially detect novel systemic biomarkers. We performed a multiplexed quantitative proteomics analysis of HDLs isolated from plasma of AAA patients (N = 14) and control study participants (N = 7). Validation was performed by western-blot (HDL), immunohistochemistry (tissue), and ELISA (plasma). HDL from AAA patients showed elevated expression of peroxiredoxin-6 (PRDX6), HLA class I histocompatibility antigen (HLA-I), retinol-binding protein 4, and paraoxonase/arylesterase 1 (PON1), whereas α-2 macroglobulin and C4b-binding protein were decreased. The main pathways associated with HDL alterations in AAA were oxidative stress and immune-inflammatory responses. In AAA tissue, PRDX6 colocalized with neutrophils, vascular smooth muscle cells, and lipid oxidation. Moreover, plasma PRDX6 was higher in AAA (N = 47) than in controls (N = 27), reflecting increased systemic oxidative stress. Finally, a positive correlation was recorded between PRDX6 and AAA diameter. The analysis of the HDL proteome demonstrates that redox imbalance is a major mechanism in AAA, identifying the antioxidant PRDX6 as a novel systemic biomarker of AAA. PMID:27934969
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Qiao, Jianjun; Wang, Jiangxin; Chen, Lei; Tian, Xiaoxu; Huang, Siqiang; Ren, Xiaoyue; Zhang, Weiwen
2012-11-02
Recent progress in metabolic engineering has led to autotrophic production of ethanol in various cyanobacterial hosts. However, cyanobacteria are known to be sensitive to ethanol, which restricts further efforts to increase ethanol production levels in these renewable host systems. To understand the mechanisms of ethanol tolerance so that engineering more robust cyanobacterial hosts can be possible, in this study, the responses of model cyanobacterial Synechocystis sp. PCC 6803 to ethanol were determined using a quantitative proteomics approach with iTRAQ LC-MS/MS technologies. The resulting high-quality proteomic data set consisted of 24,887 unique peptides corresponding to 1509 identified proteins, a coverage of approximately 42% of the predicted proteins in the Synechocystis genome. Using a cutoff of 1.5-fold change and a p-value less than 0.05, 135 and 293 unique proteins with differential abundance levels were identified between control and ethanol-treated samples at 24 and 48 h, respectively. Functional analysis showed that the Synechocystis cells employed a combination of induced common stress response, modifications of cell membrane and envelope, and induction of multiple transporters and cell mobility-related proteins as protection mechanisms against ethanol toxicity. Interestingly, our proteomic analysis revealed that proteins related to multiple aspects of photosynthesis were up-regulated in the ethanol-treated Synechocystis cells, consistent with increased chlorophyll a concentration in the cells upon ethanol exposure. The study provided the first comprehensive view of the complicated molecular mechanisms against ethanol stress and also provided a list of potential gene targets for further engineering ethanol tolerance in Synechocystis PCC 6803.
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2014-01-01
Background Hepatic resection is the preferred treatment for huge hepatocellular carcinoma (>10 cm in diameter; H-HCC). However, the patients with H-HCC suffer from poor prognosis due to the early recurrence/metastasis. The underlying mechanism of H-HCC’s early recurrence/metastasis is currently not well understood. Results Here, we describe an Isobaric Tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics approach to analyze the early recurrence/metastasis related proteins of H-HCC after radical resection through multidimensional chromatography coupled with tandem mass spectrometry (2DLC-MS/MS). The different protein expression profiles between the early recurrence/metastasis within 6 months(R/M≤6months) and late recurrence/metastasis within 6–12 months after surgery (R/M6-12months) were confirmed and might reveal different underlying molecular mechanisms. We identified 44 and 49 significantly differentially expressed proteins in the R/M≤6months group and the R/M6-12months group compared to the group who had no recurrence within 2 years post surgery (the NR/M group), respectively. Moreover, among those proteins, S100A12 and AMACR were down regulated in the R/M≤6months group but up-regulated in the R/M6-12months group; and this regulation was further confirmed in mRNA and protein level by Q-PCR, Western-Blot and Immunohistochemistry (IHC). Conclusions This current study presents the first proteomic profile of the early recurrence/metastasis of H-HCC. The results suggest that S100A12 and AMACR might be potential prognostic markers for predicting the early recurrence/metastasis of H-HCC after hepatectomy. PMID:24839399
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Zhu, Ying; Piehowski, Paul D; Zhao, Rui; Chen, Jing; Shen, Yufeng; Moore, Ronald J; Shukla, Anil K; Petyuk, Vladislav A; Campbell-Thompson, Martha; Mathews, Clayton E; Smith, Richard D; Qian, Wei-Jun; Kelly, Ryan T
2018-02-28
Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.
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Yan, Junjie; Tian, Han; Wang, Shuzhen; Shao, Jinzhen; Zheng, Yinzhen; Zhang, Hongyuan; Guo, Lin; Ding, Yi
2014-08-28
Cytoplasmic male sterility (CMS) is a widely observed phenomenon, which is especially useful in hybrid seed production. Meixiang A (MxA) is a new rice CMS line derived from a pollen-free sterile line named Yunnan ZidaoA (ZD-CMS). In this study, a homologous WA352 gene with variation in two nucleotides was identified in MxA. Cytological analysis revealed that MxA was aborted in the early uninucleate stage. The protein expression profiles of MxA and its maintainer line MeixiangB (MxB) were systematically compared using iTRAQ-based quantitative proteomics technology using young florets at the early uninucleate stage. A total of 688 proteins were quantified in both rice lines, and 45 of these proteins were found to be differentially expressed. Bioinformatics analysis indicated a large number of the proteins involved in carbohydrate metabolism or the stress response were downregulated in MxA, suggesting that these metabolic processes had been hindered during pollen development in MxA. The ROS (reactive oxygen species) level was increased in the mitochondrion of MxA, and further ultrastructural analysis showed the mitochondria with disrupted cristae in the rice CMS line MxA. These findings substantially contribute to our knowledge of pollen developmental defects in ZD-CMS rice line. MeixiangA (MxA) is a new type of rice CMS line, which is derived from pollen-free sterile line Yunnan ZidaoA. In this study, the cytological, molecular and proteomic approaches were used to study the characteristics of this new CMS line. Cytological study indicates the CMS line is aborted at the early uninucleate stage. A potential sterile gene ZD352 is identified in MxA, the protein product of which is mainly accumulated at the MMC/Meiotic stage. iTRAQ based proteomic analysis is performed to study the relevant proteins involved in the CMS occurance, 45 proteins are found to be significant differentially expressed and these proteins are involved in many cellular processes such as
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A unique proteomic profile on surface IgM ligation in unmutated chronic lymphocytic leukemia
Perrot, Aurore; Pionneau, Cédric; Nadaud, Sophie; Davi, Frédéric; Leblond, Véronique; Jacob, Frédéric; Merle-Béral, Hélène; Herbrecht, Raoul; Béné, Marie-Christine; Gribben, John G.; Vallat, Laurent
2011-01-01
Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course with 2 extreme subsets: indolent, ZAP70− and mutated immunoglobulin heavy chain gene (M-CLL); and aggressive, ZAP70+ and unmutated immunoglobulin heavy chain (UM-CLL). Given the long-term suspicion of antigenic stimulation as a primum movens in the disease, the role of the B-cell receptor has been extensively studied in various experimental settings; albeit scarcely in a comparative dynamic proteomic approach. Here we use a quantitative 2-dimensional fluorescence difference gel electrophoresis technology to compare 48 proteomic profiles of the 2 CLL subsets before and after anti-IgM ligation. Differentially expressed proteins were subsequently identified by mass spectrometry. We show that unstimulated M- and UM-CLL cells display distinct proteomic profiles. Furthermore, anti-IgM stimulation induces a specific proteomic response, more pronounced in the more aggressive CLL. Statistical analyses demonstrate several significant protein variations according to stimulation conditions. Finally, we identify an intermediate form of M-CLL cells, with an indolent profile (ZAP70−) but sharing aggressive proteomic profiles alike UM-CLL cells. Collectively, this first quantitative and dynamic proteome analysis of CLL further dissects the complex molecular pathway after B-cell receptor stimulation and depicts distinct proteomic profiles, which could lead to novel molecular stratification of the disease. PMID:21602524
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Lee, Chia-Fang; Paull, Tanya T; Person, Maria D
2013-10-04
Reactive oxygen species (ROS) play an important role in normal biological functions and pathological processes. ROS is one of the driving forces for oxidizing proteins, especially on cysteine thiols. The labile, transient, and dynamic nature of oxidative modifications poses enormous technical challenges for both accurate modification site determination and quantitation of cysteine thiols. The present study describes a mass spectrometry-based approach that allows effective discovery and quantification of irreversible cysteine modifications. The utilization of a long reverse phase column provides high-resolution chromatography to separate different forms of modified cysteine thiols from protein complexes or cell lysates. This Fourier transform mass spectrometry (FT-MS) approach enabled detection and quantitation of ataxia telangiectasia mutated (ATM) complex cysteine sulfoxidation states using Skyline MS1 filtering. When we applied the long column ultra high pressure liquid chromatography (UPLC)-MS/MS analysis, 61 and 44 peptides from cell lysates and cells were identified with cysteine modifications in response to in vitro and in vivo H2O2 oxidation, respectively. Long column ultra high pressure liquid chromatography pseudo selected reaction monitoring (UPLC-pSRM) was then developed to monitor the oxidative level of cysteine thiols in cell lysate under varying concentrations of H2O2 treatment. From UPLC-pSRM analysis, the dynamic conversion of sulfinic (S-O2H) and sulfonic acid (S-O3H) was observed within nucleoside diphosphate kinase (Nm23-H1) and heat shock 70 kDa protein 8 (Hsc70). These methods are suitable for proteome-wide studies, providing a highly sensitive, straightforward approach to identify proteins containing redox-sensitive cysteine thiols in biological systems.
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Ren, Weibo; Hou, Xiangyang; Wang, Yuqing; Badgery, Warwick; Li, Xiliang; Ding, Yong; Guo, Huiqin; Wu, Zinian; Hu, Ningning; Kong, Lingqi; Chang, Chun; Jiang, Chao; Zhang, Jize
2016-01-01
The degradation of the steppe of Inner Mongolia, due to overgrazing, has resulted in ecosystem damage as well as extensive reductions in sheep production. The growth performance of sheep is greatly reduced because of overgrazing, which triggers massive economic losses every year. The liver is an essential organ that has very important roles in multiple functions, such as nutrient metabolism, immunity and others, which are closely related to animal growth. However, to our knowledge, no detailed studies have evaluated hepatic metabolism adaption in sheep due to overgrazing. The molecular mechanisms that underlie these effects remain unclear. In the present study, our group applied isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis to investigate changes in the protein profiles of sheep hepatic tissues when nutrition was reduced due to overgrazing (12.0 sheep/ha), with the goal of characterizing the molecular mechanisms of hepatic metabolism adaption in sheep in an overgrazing condition. The body weight daily gain of sheep was greatly decreased due to overgrazing. Overall, 41 proteins were found to be differentially abundant in the hepatic tissue between a light grazing group and an overgrazing group. Most of the differentially expressed proteins identified are involved in protein metabolism, transcriptional and translational regulation, and immune response. In particular, the altered abundance of kynureninase (KYNU) and HAL (histidine ammonia-lyase) involved in protein metabolic function, integrated with the changes of serum levels of blood urea nitrogen (BUN) and glucose (GLU), suggest that overgrazing triggers a shift in energy resources from carbohydrates to proteins, causing poorer nitrogen utilization efficiency. Altogether, these results suggest that the reductions in animal growth induced by overgrazing are associated with liver proteomic changes, especially the proteins involved in nitrogen compounds metabolism
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Comparative evaluation of saliva collection methods for proteome analysis.
Golatowski, Claas; Salazar, Manuela Gesell; Dhople, Vishnu Mukund; Hammer, Elke; Kocher, Thomas; Jehmlich, Nico; Völker, Uwe
2013-04-18
Saliva collection devices are widely used for large-scale screening approaches. This study was designed to compare the suitability of three different whole-saliva collection approaches for subsequent proteome analyses. From 9 young healthy volunteers (4 women and 5 men) saliva samples were collected either unstimulated by passive drooling or stimulated using a paraffin gum or Salivette® (cotton swab). Saliva volume, protein concentration and salivary protein patterns were analyzed comparatively. Samples collected using paraffin gum showed the highest saliva volume (4.1±1.5 ml) followed by Salivette® collection (1.8±0.4 ml) and drooling (1.0±0.4 ml). Saliva protein concentrations (average 1145 μg/ml) showed no significant differences between the three sampling schemes. Each collection approach facilitated the identification of about 160 proteins (≥2 distinct peptides) per subject, but collection-method dependent variations in protein composition were observed. Passive drooling, paraffin gum and Salivette® each allows similar coverage of the whole saliva proteome, but the specific proteins observed depended on the collection approach. Thus, only one type of collection device should be used for quantitative proteome analysis in one experiment, especially when performing large-scale cross-sectional or multi-centric studies. Copyright © 2013 Elsevier B.V. All rights reserved.
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Taleb, Raghda Saad Zaghloul; Moez, Pacint; Younan, Doreen; Eisenacher, Martin; Tenbusch, Matthias; Sitek, Barbara; Bracht, Thilo
2017-12-01
Hepatocellular carcinoma (HCC) is the most common primary malignant liver tumor and a leading cause of cancer-related deaths worldwide. Cirrhosis induced by hepatitis-C virus (HCV) infection is the most critical risk factor for HCC. However, the mechanism of HCV-induced carcinogenesis is not fully understood. Plasma microparticles (PMP) contribute to numerous physiological and pathological processes and contain proteins whose composition correlates to the respective pathophysiological conditions. We analyzed PMP from 22 HCV-induced cirrhosis patients, 16 HCV-positive HCC patients with underlying cirrhosis and 18 healthy controls. PMP were isolated using ultracentrifugation and analyzed via label-free LC-MS/MS. We identified 840 protein groups and quantified 507 proteins. 159 proteins were found differentially abundant between the three experimental groups. PMP in both disease entities displayed remarkable differences in the proteome composition compared to healthy controls. Conversely, the proteome difference between both diseases was minimal. GO analysis revealed that PMP isolated from both diseases were significantly enriched in proteins involved in complement activation, while endopeptidase activity was downregulated exclusively in HCC patients. This study reports for the first time a quantitative proteome analysis for PMP from patients with HCV-induced cirrhosis and HCC. Data are available via ProteomeXchange with identifier PXD005777. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Pang, Zhili; Srivastava, Vaibhav; Liu, Xili; Bulone, Vincent
2017-04-01
The oomycete Phytophthora capsici is a plant pathogen responsible for important losses to vegetable production worldwide. Its asexual reproduction plays an important role in the rapid propagation and spread of the disease in the field. A global proteomics study was conducted to compare two key asexual life stages of P. capsici, i.e. the mycelium and cysts, to identify stage-specific biochemical processes. A total of 1200 proteins was identified using qualitative and quantitative proteomics. The transcript abundance of some of the enriched proteins was also analysed by quantitative real-time polymerase chain reaction. Seventy-three proteins exhibited different levels of abundance between the mycelium and cysts. The proteins enriched in the mycelium are mainly associated with glycolysis, the tricarboxylic acid (or citric acid) cycle and the pentose phosphate pathway, providing the energy required for the biosynthesis of cellular building blocks and hyphal growth. In contrast, the proteins that are predominant in cysts are essentially involved in fatty acid degradation, suggesting that the early infection stage of the pathogen relies primarily on fatty acid degradation for energy production. The data provide a better understanding of P. capsici biology and suggest potential metabolic targets at the two different developmental stages for disease control. © 2016 BSPP AND JOHN WILEY & SONS LTD.
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Plant proteome analysis: a 2006 update.
Jorrín, Jesús V; Maldonado, Ana M; Castillejo, Ma Angeles
2007-08-01
This 2006 'Plant Proteomics Update' is a continuation of the two previously published in 'Proteomics' by 2004 (Canovas et al., Proteomics 2004, 4, 285-298) and 2006 (Rossignol et al., Proteomics 2006, 6, 5529-5548) and it aims to bring up-to-date the contribution of proteomics to plant biology on the basis of the original research papers published throughout 2006, with references to those appearing last year. According to the published papers and topics addressed, we can conclude that, as observed for the three previous years, there has been a quantitative, but not qualitative leap in plant proteomics. The full potential of proteomics is far from being exploited in plant biology research, especially if compared to other organisms, mainly yeast and humans, and a number of challenges, mainly technological, remain to be tackled. The original papers published last year numbered nearly 100 and deal with the proteome of at least 26 plant species, with a high percentage for Arabidopsis thaliana (28) and rice (11). Scientific objectives ranged from proteomic analysis of organs/tissues/cell suspensions (57) or subcellular fractions (29), to the study of plant development (12), the effect of hormones and signalling molecules (8) and response to symbionts (4) and stresses (27). A small number of contributions have covered PTMs (8) and protein interactions (4). 2-DE (specifically IEF-SDS-PAGE) coupled to MS still constitutes the almost unique platform utilized in plant proteome analysis. The application of gel-free protein separation methods and 'second generation' proteomic techniques such as multidimensional protein identification technology (MudPIT), and those for quantitative proteomics including DIGE, isotope-coded affinity tags (ICAT), iTRAQ and stable isotope labelling by amino acids in cell culture (SILAC) still remains anecdotal. This review is divided into seven sections: Introduction, Methodology, Subcellular proteomes, Development, Responses to biotic and abiotic
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A Scalable Approach for Protein False Discovery Rate Estimation in Large Proteomic Data Sets.
Savitski, Mikhail M; Wilhelm, Mathias; Hahne, Hannes; Kuster, Bernhard; Bantscheff, Marcus
2015-09-01
Calculating the number of confidently identified proteins and estimating false discovery rate (FDR) is a challenge when analyzing very large proteomic data sets such as entire human proteomes. Biological and technical heterogeneity in proteomic experiments further add to the challenge and there are strong differences in opinion regarding the conceptual validity of a protein FDR and no consensus regarding the methodology for protein FDR determination. There are also limitations inherent to the widely used classic target-decoy strategy that particularly show when analyzing very large data sets and that lead to a strong over-representation of decoy identifications. In this study, we investigated the merits of the classic, as well as a novel target-decoy-based protein FDR estimation approach, taking advantage of a heterogeneous data collection comprised of ∼19,000 LC-MS/MS runs deposited in ProteomicsDB (https://www.proteomicsdb.org). The "picked" protein FDR approach treats target and decoy sequences of the same protein as a pair rather than as individual entities and chooses either the target or the decoy sequence depending on which receives the highest score. We investigated the performance of this approach in combination with q-value based peptide scoring to normalize sample-, instrument-, and search engine-specific differences. The "picked" target-decoy strategy performed best when protein scoring was based on the best peptide q-value for each protein yielding a stable number of true positive protein identifications over a wide range of q-value thresholds. We show that this simple and unbiased strategy eliminates a conceptual issue in the commonly used "classic" protein FDR approach that causes overprediction of false-positive protein identification in large data sets. The approach scales from small to very large data sets without losing performance, consistently increases the number of true-positive protein identifications and is readily implemented in
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Huang, Xin; Tian, Changhai; Liu, Miao
2012-04-06
Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. Using thismore » platform a total of 2,481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1) and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.« less
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Xu, Benhong; Gao, Yanpan; Zhan, Shaohua; Ge, Wei
2017-07-01
Lysosomes play vital roles in both innate and adaptive immunity. It is widely accepted that lysosomes do not function exclusively as a digestive organelle. It is also involved in the process of immune cells against pathogens. However, the changes in the lysosomal proteome caused by infection with various microbes are still largely unknown, and our understanding of the proteome of the purified lysosome is another obstacle that needs to be resolved. Here, we performed a proteomic study on lysosomes enriched from THP1 cells after infection with Listeria monocytogenes (L.m), Herpes Simplex Virus 1 (HSV-1) and Vesicular Stomatitis Virus (VSV). In combination with the gene ontology (GO) analysis, we identified 284 lysosomal-related proteins from a total of 4560 proteins. We also constructed the protein-protein interaction networks for the differentially expressed proteins and revealed the core lysosomal proteins, including SRC in the L. m treated group, SRC, GLB1, HEXA and HEXB in the HSV-1 treated group and GLB1, CTSA, CTSB, HEXA and HEXB in the VSV treated group, which are involved in responding to diverse microbial infections. This study not only reveals variable lysosome responses depending on the bacterial or virus infection, but also provides the evidence based on which we propose a novel approach to proteome research for investigation of the function of the enriched organelles. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Screening Novel Molecular Targets of Metformin in Breast Cancer by Proteomic Approach
Al-Zaidan, Lobna; El Ruz, Rasha Abu; Malki, Ahmed M.
2017-01-01
Metformin is a commonly prescribed antihyperglycemic drug, and has been investigated in vivo and in vitro for its effect to improve the comorbidity of diabetes and various types of cancers. Several studies investigated the therapeutic mechanisms of metformin on cancer cells, but the exact mechanism of metformin’s effect on the proteomic pathways of cancer cells is yet to be further investigated. The main objective of our research line is to discover safe and alternative therapeutic options for breast cancer, we aimed in this study to design a novel “bottom up proteomics workflow” in which proteins were first broken into peptides to reveal their identity, then the proteomes were precisely evaluated using spectrometry analysis. In our study, metformin suppressed cell proliferation and induced apoptosis in human breast carcinoma cell line MCF-7 with minimal toxicity to normal breast epithelial cells MCF-10. Metformin induced apoptosis by arresting cells in G1 phase as evaluated by flow cytometric analysis. Moreover, The G1 phase arrest for the MCF-7 has been confirmed by increased expression levels of p21 and reduction in cyclin D1 level. Additionally, metformin increased the expression levels of p53, Bax, Bad while it reduced expression levels of Akt, Bcl-2, and Mdm2. The study employed a serviceable strategy that investigates metformin-dependent changes in the proteome using a literature-derived network. The protein extracts of the treated and untreated cell lines were analyzed employing proteomic approaches; the findings conveyed a proposed mechanism of the effectual tactics of metformin on breast cancer cells. Metformin proposed an antibreast cancer effect through the examination of the proteomic pathways upon the MCF-7 and MCF-10A exposure to the drug. Our findings proposed prolific proteomic changes that revealed the therapeutic mechanisms of metformin on breast cancer cells upon their exposure. In conclusion, the reported proteomic pathways lead to
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Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics.
Bennett, Eric J; Rush, John; Gygi, Steven P; Harper, J Wade
2010-12-10
Dynamic reorganization of signaling systems frequently accompanies pathway perturbations, yet quantitative studies of network remodeling by pathway stimuli are lacking. Here, we report the development of a quantitative proteomics platform centered on multiplex absolute quantification (AQUA) technology to elucidate the architecture of the cullin-RING ubiquitin ligase (CRL) network and to evaluate current models of dynamic CRL remodeling. Current models suggest that CRL complexes are controlled by cycles of CRL deneddylation and CAND1 binding. Contrary to expectations, acute CRL inhibition with MLN4924, an inhibitor of the NEDD8-activating enzyme, does not result in a global reorganization of the CRL network. Examination of CRL complex stoichiometry reveals that, independent of cullin neddylation, a large fraction of cullins are assembled with adaptor modules, whereas only a small fraction are associated with CAND1. These studies suggest an alternative model of CRL dynamicity where the abundance of adaptor modules, rather than cycles of neddylation and CAND1 binding, drives CRL network organization. Copyright © 2010 Elsevier Inc. All rights reserved.
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Song, Hao; Wang, Hai-Yan; Zhang, Tao
2016-06-15
Larval metamorphosis of the veined rapa whelk (Rapana venosa) is a pelagic to benthic transition that involves considerable structural and physiological changes. Because metamorphosis plays a pivotal role in R. venosa commercial breeding and natural populations, the endogenous proteins that drive this transition attract considerable interest. This study is the first to perform a comprehensive and quantitative proteomic analysis related to metamorphosis in a marine gastropod. We analyzed the proteomes of competent R. venosa larvae and post-larvae, resulting in the identification of 5312 proteins, including 470 that were downregulated and 668 that were upregulated after metamorphosis. The differentially expressed proteins reflected multiple processes involved in metamorphosis, including cytoskeleton and cell adhesion, ingestion and digestion, stress response and immunity, as well as specific tissue development. Our data improve understanding of the physiological traits controlling R. venosa metamorphosis and provide a solid basis for further study.
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Advanced Mass Spectrometric Methods for the Rapid and Quantitative Characterization of Proteomes
Smith, Richard D.
2002-01-01
Progress is reviewedmore » towards the development of a global strategy that aims to extend the sensitivity, dynamic range, comprehensiveness and throughput of proteomic measurements based upon the use of high performance separations and mass spectrometry. The approach uses high accuracy mass measurements from Fourier transform ion cyclotron resonance mass spectrometry (FTICR) to validate peptide ‘accurate mass tags’ (AMTs) produced by global protein enzymatic digestions for a specific organism, tissue or cell type from ‘potential mass tags’ tentatively identified using conventional tandem mass spectrometry (MS/MS). This provides the basis for subsequent measurements without the need for MS/ MS. High resolution capillary liquid chromatography separations combined with high sensitivity, and high resolution accurate FTICR measurements are shown to be capable of characterizing peptide mixtures of more than 10 5 components. The strategy has been initially demonstrated using the microorganisms Saccharomyces cerevisiae and Deinococcus radiodurans. Advantages of the approach include the high confidence of protein identification, its broad proteome coverage, high sensitivity, and the capability for stableisotope labeling methods for precise relative protein abundance measurements. Abbreviations : LC, liquid chromatography; FTICR, Fourier transform ion cyclotron resonance; AMT, accurate mass tag; PMT, potential mass tag; MMA, mass measurement accuracy; MS, mass spectrometry; MS/MS, tandem mass spectrometry; ppm, parts per million.« less
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Cilia, M.; Fish, T.; Yang, X.; Mclaughlin, M.; Thannhauser, T. W.
2009-01-01
Protein extraction methods can vary widely in reproducibility and in representation of the total proteome, yet there are limited data comparing protein isolation methods. The methodical comparison of protein isolation methods is the first critical step for proteomic studies. To address this, we compared three methods for isolation, purification, and solubilization of insect proteins. The aphid Schizaphis graminum, an agricultural pest, was the source of insect tissue. Proteins were extracted using TCA in acetone (TCA-acetone), phenol, or multi-detergents in a chaotrope solution. Extracted proteins were solubilized in a multiple chaotrope solution and examined using 1-D and 2-D electrophoresis and compared directly using 2-D Difference Gel Electrophoresis (2-D DIGE). Mass spectrometry was used to identify proteins from each extraction type. We were unable to ascribe the differences in the proteins extracted to particular physical characteristics, cell location, or biological function. The TCA-acetone extraction yielded the greatest amount of protein from aphid tissues. Each extraction method isolated a unique subset of the aphid proteome. The TCA-acetone method was explored further for its quantitative reliability using 2-D DIGE. Principal component analysis showed that little of the variation in the data was a result of technical issues, thus demonstrating that the TCA-acetone extraction is a reliable method for preparing aphid proteins for a quantitative proteomics experiment. These data suggest that although the TCA-acetone method is a suitable method for quantitative aphid proteomics, a combination of extraction approaches is recommended for increasing proteome coverage when using gel-based separation techniques. PMID:19721822
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Knudsen, Anders Dahl; Bennike, Tue; Kjeldal, Henrik; Birkelund, Svend; Otzen, Daniel Erik; Stensballe, Allan
2014-05-30
We describe Condenser, a freely available, comprehensive open-source tool for merging multidimensional quantitative proteomics data from the Matrix Science Mascot Distiller Quantitation Toolbox into a common format ready for subsequent bioinformatic analysis. A number of different relative quantitation technologies, such as metabolic (15)N and amino acid stable isotope incorporation, label-free and chemical-label quantitation are supported. The program features multiple options for curative filtering of the quantified peptides, allowing the user to choose data quality thresholds appropriate for the current dataset, and ensure the quality of the calculated relative protein abundances. Condenser also features optional global normalization, peptide outlier removal, multiple testing and calculation of t-test statistics for highlighting and evaluating proteins with significantly altered relative protein abundances. Condenser provides an attractive addition to the gold-standard quantitative workflow of Mascot Distiller, allowing easy handling of larger multi-dimensional experiments. Source code, binaries, test data set and documentation are available at http://condenser.googlecode.com/. Copyright © 2014 Elsevier B.V. All rights reserved.
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Quality Assessments of Long-Term Quantitative Proteomic Analysis of Breast Cancer Xenograft Tissues
DOE Office of Scientific and Technical Information (OSTI.GOV)
Zhou, Jian-Ying; Chen, Lijun; Zhang, Bai
The identification of protein biomarkers requires large-scale analysis of human specimens to achieve statistical significance. In this study, we evaluated the long-term reproducibility of an iTRAQ (isobaric tags for relative and absolute quantification) based quantitative proteomics strategy using one channel for universal normalization across all samples. A total of 307 liquid chromatography tandem mass spectrometric (LC-MS/MS) analyses were completed, generating 107 one-dimensional (1D) LC-MS/MS datasets and 8 offline two-dimensional (2D) LC-MS/MS datasets (25 fractions for each set) for human-in-mouse breast cancer xenograft tissues representative of basal and luminal subtypes. Such large-scale studies require the implementation of robust metrics to assessmore » the contributions of technical and biological variability in the qualitative and quantitative data. Accordingly, we developed a quantification confidence score based on the quality of each peptide-spectrum match (PSM) to remove quantification outliers from each analysis. After combining confidence score filtering and statistical analysis, reproducible protein identification and quantitative results were achieved from LC-MS/MS datasets collected over a 16 month period.« less
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Proteomic dataset of the sea urchin Paracentrotus lividus adhesive organs and secreted adhesive.
Lebesgue, Nicolas; da Costa, Gonçalo; Ribeiro, Raquel Mesquita; Ribeiro-Silva, Cristina; Martins, Gabriel G; Matranga, Valeria; Scholten, Arjen; Cordeiro, Carlos; Heck, Albert J R; Santos, Romana
2016-06-01
Sea urchins have specialized adhesive organs called tube feet, which mediate strong but reversible adhesion. Tube feet are composed by a disc, producing adhesive and de-adhesive secretions for substratum attachment, and a stem for movement. After detachment the secreted adhesive remains bound to the substratum as a footprint. Recently, a label-free quantitative proteomic approach coupled with the latest mass-spectrometry technology was used to analyze the differential proteome of Paracentrotus lividus adhesive organ, comparing protein expression levels in the tube feet adhesive part (the disc) versus the non-adhesive part (the stem), and also to profile the proteome of the secreted adhesive (glue). This data article contains complementary figures and results related to the research article "Deciphering the molecular mechanisms underlying sea urchin reversible adhesion: a quantitative proteomics approach" (Lebesgue et al., 2016) [1]. Here we provide a dataset of 1384 non-redundant proteins, their fragmented peptides and expression levels, resultant from the analysis of the tube feet differential proteome. Of these, 163 highly over-expressed tube feet disc proteins (>3-fold), likely representing the most relevant proteins for sea urchin reversible adhesion, were further annotated in order to determine the potential functions. In addition, we provide a dataset of 611 non-redundant proteins identified in the secreted adhesive proteome, as well as their functional annotation and grouping in 5 major protein groups related with adhesive exocytosis, and microbial protection. This list was further analyzed to identify the most abundant protein groups and pinpoint putative adhesive proteins, such as Nectin, the most abundant adhesive protein in sea urchin glue. The obtained data uncover the key proteins involved in sea urchins reversible adhesion, representing a step forward to the development of new wet-effective bio-inspired adhesives.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Zhu, Ying; Piehowski, Paul D.; Zhao, Rui
Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here in this paper, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Betweenmore » Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.« less
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Zhu, Ying; Piehowski, Paul D.; Zhao, Rui; ...
2018-02-28
Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here in this paper, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Betweenmore » Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Mu, Jun; Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing; Chongqing Key Laboratory of Neurobiology, Chongqing
Purpose: Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). Methods: CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology andmore » proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Results: Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. Conclusions: CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. - Highlights: • The first proteomic study on the cerebrospinal fluid of tuberculous meningitis patients using iTRAQ. • Identify 4 differential proteins invloved in the lipid metabolism. • Elevated expression of Apo
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Design and analysis of quantitative differential proteomics investigations using LC-MS technology.
Bukhman, Yury V; Dharsee, Moyez; Ewing, Rob; Chu, Peter; Topaloglou, Thodoros; Le Bihan, Thierry; Goh, Theo; Duewel, Henry; Stewart, Ian I; Wisniewski, Jacek R; Ng, Nancy F
2008-02-01
Liquid chromatography-mass spectrometry (LC-MS)-based proteomics is becoming an increasingly important tool in characterizing the abundance of proteins in biological samples of various types and across conditions. Effects of disease or drug treatments on protein abundance are of particular interest for the characterization of biological processes and the identification of biomarkers. Although state-of-the-art instrumentation is available to make high-quality measurements and commercially available software is available to process the data, the complexity of the technology and data presents challenges for bioinformaticians and statisticians. Here, we describe a pipeline for the analysis of quantitative LC-MS data. Key components of this pipeline include experimental design (sample pooling, blocking, and randomization) as well as deconvolution and alignment of mass chromatograms to generate a matrix of molecular abundance profiles. An important challenge in LC-MS-based quantitation is to be able to accurately identify and assign abundance measurements to members of protein families. To address this issue, we implement a novel statistical method for inferring the relative abundance of related members of protein families from tryptic peptide intensities. This pipeline has been used to analyze quantitative LC-MS data from multiple biomarker discovery projects. We illustrate our pipeline here with examples from two of these studies, and show that the pipeline constitutes a complete workable framework for LC-MS-based differential quantitation. Supplementary material is available at http://iec01.mie.utoronto.ca/~thodoros/Bukhman/.
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Clinical proteomic analysis of scrub typhus infection.
Park, Edmond Changkyun; Lee, Sang-Yeop; Yun, Sung Ho; Choi, Chi-Won; Lee, Hayoung; Song, Hyun Seok; Jun, Sangmi; Kim, Gun-Hwa; Lee, Chang-Seop; Kim, Seung Il
2018-01-01
Scrub typhus is an acute and febrile infectious disease caused by the Gram-negative α-proteobacterium Orientia tsutsugamushi from the family Rickettsiaceae that is widely distributed in Northern, Southern and Eastern Asia. In the present study, we analysed the serum proteome of scrub typhus patients to investigate specific clinical protein patterns in an attempt to explain pathophysiology and discover potential biomarkers of infection. Serum samples were collected from three patients (before and after treatment with antibiotics) and three healthy subjects. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry was performed to identify differentially abundant proteins using quantitative proteomic approaches. Bioinformatic analysis was then performed using Ingenuity Pathway Analysis. Proteomic analysis identified 236 serum proteins, of which 32 were differentially expressed in normal subjects, naive scrub typhus patients and patients treated with antibiotics. Comparative bioinformatic analysis of the identified proteins revealed up-regulation of proteins involved in immune responses, especially complement system, following infection with O. tsutsugamushi , and normal expression was largely rescued by antibiotic treatment. This is the first proteomic study of clinical serum samples from scrub typhus patients. Proteomic analysis identified changes in protein expression upon infection with O. tsutsugamushi and following antibiotic treatment. Our results provide valuable information for further investigation of scrub typhus therapy and diagnosis.
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Yang, Wei; Kim, Yongsoo; Kim, Taek-Kyun; Keay, Susan K; Kim, Kwang Pyo; Steen, Hanno; Freeman, Michael R; Hwang, Daehee; Kim, Jayoung
2012-12-01
identify more differentially expressed genes with a lower false discovery rate from a previously published microarray data set, an integrative hypothesis-testing statistical approach was applied. • For validation experiments, expression and phosphorylation levels of select proteins were evaluated by western blotting. • Integration analysis of this transcriptomics data set with our own quantitative proteomics data set identified 10 genes that are potentially regulated by APF in vivo from 4140 differentially expressed genes identified with a false discovery rate of 1%. • Of these, five (i.e. JUP, MAPKSP1, GSPT1, PTGS2/COX-2 and XPOT) were found to be prominent after network modelling of the common genes identified in the proteomics and microarray studies. • This molecular signature reflects the biological processes of cell adhesion, cell proliferation and inflammation, which is consistent with the known physiological effects of APF. • Lastly, we found the mammalian target of rapamycin pathway was down-regulated in response to APF. • This unbiased integration analysis of in vitro quantitative proteomics data with in vivo quantitative transcriptomics data led to the identification of potential downstream mediators of the APF signal transduction pathway. © 2012 THE AUTHORS. BJU INTERNATIONAL © 2012 BJU INTERNATIONAL.
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Mudaliar, Manikhandan; Tassi, Riccardo; Thomas, Funmilola C; McNeilly, Tom N; Weidt, Stefan K; McLaughlin, Mark; Wilson, David; Burchmore, Richard; Herzyk, Pawel; Eckersall, P David; Zadoks, Ruth N
2016-08-16
Mastitis, inflammation of the mammary gland, is the most common and costly disease of dairy cattle in the western world. It is primarily caused by bacteria, with Streptococcus uberis as one of the most prevalent causative agents. To characterize the proteome during Streptococcus uberis mastitis, an experimentally induced model of intramammary infection was used. Milk whey samples obtained from 6 cows at 6 time points were processed using label-free relative quantitative proteomics. This proteomic analysis complements clinical, bacteriological and immunological studies as well as peptidomic and metabolomic analysis of the same challenge model. A total of 2552 non-redundant bovine peptides were identified, and from these, 570 bovine proteins were quantified. Hierarchical cluster analysis and principal component analysis showed clear clustering of results by stage of infection, with similarities between pre-infection and resolution stages (0 and 312 h post challenge), early infection stages (36 and 42 h post challenge) and late infection stages (57 and 81 h post challenge). Ingenuity pathway analysis identified upregulation of acute phase protein pathways over the course of infection, with dominance of different acute phase proteins at different time points based on differential expression analysis. Antimicrobial peptides, notably cathelicidins and peptidoglycan recognition protein, were upregulated at all time points post challenge and peaked at 57 h, which coincided with 10 000-fold decrease in average bacterial counts. The integration of clinical, bacteriological, immunological and quantitative proteomics and other-omic data provides a more detailed systems level view of the host response to mastitis than has been achieved previously.
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Mu, Jun; Yang, Yongtao; Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng; Xie, Peng
2015-10-30
Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. Copyright © 2015 Elsevier Inc. All rights reserved.
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Chen, Ru; Pan, Sheng; Cooke, Kelly; Moyes, Kara White; Bronner, Mary P.; Goodlett, David R.; Aebersold, Ruedi; Brentnall, Teresa A.
2008-01-01
Objectives Pancreatitis is an inflammatory condition of the pancreas. However, it often shares many molecular features with pancreatic cancer. Biomarkers present in pancreatic cancer frequently occur in the setting of pancreatitis. The efforts to develop diagnostic biomarkers for pancreatic cancer have thus been complicated by the false-positive involvement of pancreatitis. Methods In an attempt to develop protein biomarkers for pancreatic cancer, we previously use quantitative proteomics to identify and quantify the proteins from pancreatic cancer juice. Pancreatic juice is a rich source of proteins that are shed by the pancreatic ductal cells. In this study, we used a similar approach to identify and quantify proteins from pancreatitis juice. Results In total, 72 proteins were identified and quantified in the comparison of pancreatic juice from pancreatitis patients versus pooled normal control juice. Nineteen of the juice proteins were overexpressed, and 8 were underexpressed in pancreatitis juice by at least 2-fold compared with normal pancreatic juice. Of these 27 differentially expressed proteins in pancreatitis, 9 proteins were also differentially expressed in the pancreatic juice from pancreatic cancer patient. Conclusions Identification of these differentially expressed proteins from pancreatitis juice provides useful information for future study of specific pancreatitis-associated proteins and to eliminate potential false-positive biomarkers for pancreatic cancer. PMID:17198186
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Chen, Ru; Pan, Sheng; Cooke, Kelly; Moyes, Kara White; Bronner, Mary P; Goodlett, David R; Aebersold, Ruedi; Brentnall, Teresa A
2007-01-01
Pancreatitis is an inflammatory condition of the pancreas. However, it often shares many molecular features with pancreatic cancer. Biomarkers present in pancreatic cancer frequently occur in the setting of pancreatitis. The efforts to develop diagnostic biomarkers for pancreatic cancer have thus been complicated by the false-positive involvement of pancreatitis. In an attempt to develop protein biomarkers for pancreatic cancer, we previously use quantitative proteomics to identify and quantify the proteins from pancreatic cancer juice. Pancreatic juice is a rich source of proteins that are shed by the pancreatic ductal cells. In this study, we used a similar approach to identify and quantify proteins from pancreatitis juice. In total, 72 proteins were identified and quantified in the comparison of pancreatic juice from pancreatitis patients versus pooled normal control juice. Nineteen of the juice proteins were overexpressed, and 8 were underexpressed in pancreatitis juice by at least 2-fold compared with normal pancreatic juice. Of these 27 differentially expressed proteins in pancreatitis, 9 proteins were also differentially expressed in the pancreatic juice from pancreatic cancer patient. Identification of these differentially expressed proteins from pancreatitis juice provides useful information for future study of specific pancreatitis-associated proteins and to eliminate potential false-positive biomarkers for pancreatic cancer.
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Bi, Xin; Jin, Yibao; Gao, Xiang; Liu, Feng; Gao, Dan; Jiang, Yuyang; Liu, Hongxia
2013-01-01
Pokemon is a transcription regulator involved in embryonic development, cellular differentiation and oncogenesis. It is aberrantly overexpressed in multiple human cancers including Hepatocellular carcinoma (HCC) and is considered as a promising biomarker for HCC. In this work, the isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics strategy was used to investigate the proteomic profile associated with Pokemon in human HCC cell line QGY7703 and human hepatocyte line HL7702. Samples were labeled with four-plex iTRAQ reagents followed by two-dimensional liquid chromatography coupled with tandem mass spectrometry analysis. A total of 24 differentially expressed proteins were selected as significant. Nine proteins were potentially up-regulated by Pokemon while 15 proteins were potentially down-regulated and many proteins were previously identified as potential biomarkers for HCC. Gene ontology (GO) term enrichment revealed that the listed proteins were mainly involved in DNA metabolism and biosynthesis process. The changes of glucose-6-phosphate 1-dehydrogenase (G6PD, up-regulated) and ribonucleoside-diphosphate reductase large sub-unit (RIM1, down-regulated) were validated by Western blotting analysis and denoted as Pokemon's function of oncogenesis. We also found that Pokemon potentially repressed the expression of highly clustered proteins (MCM3, MCM5, MCM6, MCM7) which played key roles in promoting DNA replication. Altogether, our results may help better understand the role of Pokemon in HCC and promote the clinical applications.
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Manteca, Angel; Sanchez, Jesus; Jung, Hye R.; Schwämmle, Veit; Jensen, Ole N.
2010-01-01
Streptomyces species produce many clinically important secondary metabolites, including antibiotics and antitumorals. They have a complex developmental cycle, including programmed cell death phenomena, that makes this bacterium a multicellular prokaryotic model. There are two differentiated mycelial stages: an early compartmentalized vegetative mycelium (first mycelium) and a multinucleated reproductive mycelium (second mycelium) arising after programmed cell death processes. In the present study, we made a detailed proteomics analysis of the distinct developmental stages of solid confluent Streptomyces coelicolor cultures using iTRAQ (isobaric tags for relative and absolute quantitation) labeling and LC-MS/MS. A new experimental approach was developed to obtain homogeneous samples at each developmental stage (temporal protein analysis) and also to obtain membrane and cytosolic protein fractions (spatial protein analysis). A total of 345 proteins were quantified in two biological replicates. Comparative bioinformatics analyses revealed the switch from primary to secondary metabolism between the initial compartmentalized mycelium and the multinucleated hyphae. PMID:20224110
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Wu, Si; Brown, Joseph N.; Tolic, Nikola
There are several notable challenges inherent to fully characterizing the entirety of the human saliva proteome using bottom-up approaches, including polymorphic isoforms, post-translational modifications, unique splice variants, deletions, and truncations. To address these challenges, we have developed a top-down based liquid chromatography-mass spectrometry (LC-MS) approach, which cataloged 20 major human salivary proteins with a total of 83 proteoforms, containing a broad range of post-translational modifications. Among these proteins, several previously reported disease biomarker proteins were identified at the intact protein level, such as beta-2 microglobulin (B2M). In addition, intact glycosylated proteoforms of several saliva proteins were also characterized, including intactmore » N-glycosylated protein prolactin inducible protein (PIP) and O-glycosylated acidic protein rich protein (aPRP). These characterized proteoforms constitute an intact saliva proteoform database, which was used for quantitative comparison of intact salivary proteoforms among six healthy individuals. Human parotid (PS) and submandibular/sublingual gland (SMSL) secretion samples (2 μg of protein each) from six healthy individuals were compared using RPLC coupled with the 12T FTICR mass spectrometer. Significantly different protein and PTM patterns were resolved with high reproducibility between PS and SMSL glands. The results from this study provide further insight into the potential mechanisms of PTM pathways in oral glandular secretion, expanding our knowledge of this complex yet easily accessible fluid. Intact protein LC-MS approach presented herein can potentially be applied for rapid and accurate identification of biomarkers from only a few microliters of human glandular saliva.« less
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Zhang, Tong; Meng, Li; Kong, Wenwen; Yin, Zepeng; Wang, Yang; Schneider, Jacqueline D; Chen, Sixue
2018-03-20
Jasmonate ZIM-domain (JAZ) proteins are key transcriptional repressors regulating various biological processes. Although many studies have studied JAZ proteins by genetic and biochemical analyses, little is known about JAZ7-associated global protein networks and how JAZ7 contributes to bacterial pathogen defense. In this study, we aim to fill this knowledge gap by conducting unbiased large-scale quantitative proteomics using tandem mass tags (TMT). We compared the proteomes of a JAZ7 knock-out line, a JAZ7 overexpression line, as well as the wild type Arabidopsis plants in the presence and absence of Pseudomonas syringae DC3000 infection. Both pairwise comparison and multi-factor analysis of variance reveal that differential proteins are enriched in biological processes such as primary and secondary metabolism, redox regulation, and response to stress. The differential regulation in these pathways may account for the alterations in plant size, redox homeostasis and accumulation of glucosinolates. In addition, possible interplay between genotype and environment is suggested as the abundance of seven proteins is influenced by the interaction of the two factors. Collectively, we demonstrate a role of JAZ7 in pathogen defense and provide a list of proteins that are uniquely responsive to genetic disruption, pathogen infection, or the interaction between genotypes and environmental factors. We report proteomic changes as a result of genetic perturbation of JAZ7, and the contribution of JAZ7 in plant immunity. Specifically, the similarity between the proteomes of a JAZ7 knockout mutant and the wild type plants confirmed the functional redundancy of JAZs. In contrast, JAZ7 overexpression plants were much different, and proteomic analysis of the JAZ7 overexpression plants under Pst DC3000 infection revealed that JAZ7 may regulate plant immunity via ROS modulation, energy balance and glucosinolate biosynthesis. Multiple variate analysis for this two-factor proteomics
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Lazar, Cosmin; Gatto, Laurent; Ferro, Myriam; Bruley, Christophe; Burger, Thomas
2016-04-01
Missing values are a genuine issue in label-free quantitative proteomics. Recent works have surveyed the different statistical methods to conduct imputation and have compared them on real or simulated data sets and recommended a list of missing value imputation methods for proteomics application. Although insightful, these comparisons do not account for two important facts: (i) depending on the proteomics data set, the missingness mechanism may be of different natures and (ii) each imputation method is devoted to a specific type of missingness mechanism. As a result, we believe that the question at stake is not to find the most accurate imputation method in general but instead the most appropriate one. We describe a series of comparisons that support our views: For instance, we show that a supposedly "under-performing" method (i.e., giving baseline average results), if applied at the "appropriate" time in the data-processing pipeline (before or after peptide aggregation) on a data set with the "appropriate" nature of missing values, can outperform a blindly applied, supposedly "better-performing" method (i.e., the reference method from the state-of-the-art). This leads us to formulate few practical guidelines regarding the choice and the application of an imputation method in a proteomics context.
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Salunkhe, Vishal; De Cuyper, Iris M; Papadopoulos, Petros; van der Meer, Pieter F; Daal, Brunette B; Villa-Fajardo, María; de Korte, Dirk; van den Berg, Timo K; Gutiérrez, Laura
2018-03-19
Platelet concentrates (PCs) represent a blood transfusion product with a major concern for safety as their storage temperature (20-24°C) allows bacterial growth, and their maximum storage time period (less than a week) precludes complete microbiological testing. Pathogen inactivation technologies (PITs) provide an additional layer of safety to the blood transfusion products from known and unknown pathogens such as bacteria, viruses, and parasites. In this context, PITs, such as Mirasol Pathogen Reduction Technology (PRT), have been developed and are implemented in many countries. However, several studies have shown in vitro that Mirasol PRT induces a certain level of platelet shape change, hyperactivation, basal degranulation, and increased oxidative damage during storage. It has been suggested that Mirasol PRT might accelerate what has been described as the platelet storage lesion (PSL), but supportive molecular signatures have not been obtained. We aimed at dissecting the influence of both variables, that is, Mirasol PRT and storage time, at the proteome level. We present comprehensive proteomics data analysis of Control PCs and PCs treated with Mirasol PRT at storage days 1, 2, 6, and 8. Our workflow was set to perform proteomics analysis using a gel-free and label-free quantification (LFQ) approach. Semi-quantification was based on LFQ signal intensities of identified proteins using MaxQuant/Perseus software platform. Data are available via ProteomeXchange with identifier PXD008119. We identified marginal differences between Mirasol PRT and Control PCs during storage. However, those significant changes at the proteome level were specifically related to the functional aspects previously described to affect platelets upon Mirasol PRT. In addition, the effect of Mirasol PRT on the platelet proteome appeared not to be exclusively due to an accelerated or enhanced PSL. In summary, semi-quantitative proteomics allows to discern between proteome changes due to
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Dineshram, R; Quan, Q; Sharma, Rakesh; Chandramouli, Kondethimmanahalli; Yalamanchili, Hari Krishna; Chu, Ivan; Thiyagarajan, Vengatesen
2015-12-01
Decreasing pH due to anthropogenic CO2 inputs, called ocean acidification (OA), can make coastal environments unfavorable for oysters. This is a serious socioeconomical issue for China which supplies >70% of the world's edible oysters. Here, we present an iTRAQ-based protein profiling approach for the detection and quantification of proteome changes under OA in the early life stage of a commercially important oyster, Crassostrea hongkongensis. Availability of complete genome sequence for the pacific oyster (Crassostrea gigas) enabled us to confidently quantify over 1500 proteins in larval oysters. Over 7% of the proteome was altered in response to OA at pHNBS 7.6. Analysis of differentially expressed proteins and their associated functional pathways showed an upregulation of proteins involved in calcification, metabolic processes, and oxidative stress, each of which may be important in physiological adaptation of this species to OA. The downregulation of cytoskeletal and signal transduction proteins, on the other hand, might have impaired cellular dynamics and organelle development under OA. However, there were no significant detrimental effects in developmental processes such as metamorphic success. Implications of the differentially expressed proteins and metabolic pathways in the development of OA resistance in oyster larvae are discussed. The MS proteomics data have been deposited to the ProteomeXchange with identifiers PXD002138 (http://proteomecentral.proteomexchange.org/dataset/PXD002138). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Kuo, Kung-Kai; Kuo, Chao-Jen; Chiu, Chiang-Yen; Liang, Shih-Shin; Huang, Chun-Hao; Chi, Shu-Wen; Tsai, Kun-Bow; Chen, Chiao-Yun; Hsi, Edward; Cheng, Kuang-Hung; Chiou, Shyh-Horng
2016-01-01
Objectives The aim of this study was to identify differentially expressed proteins among various stages of pancreatic ductal adenocarcinoma (PDAC) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry and stable isotope dimethyl labeling. Methods Differentially expressed proteins were identified and compared based on the mass spectral differences of their isotope-labeled peptide fragments generated from protease digestion. Results Our quantitative proteomic analysis of the differentially expressed proteins with stable isotope (deuterium/hydrogen ratio, ≥2) identified a total of 353 proteins, with at least 5 protein biomarker proteins that were significantly differentially expressed between cancer and normal mice by at least a 2-fold alteration. These 5 protein biomarker candidates include α-enolase, α-catenin, 14-3-3 β, VDAC1, and calmodulin with high confidence levels. The expression levels were also found to be in agreement with those examined by Western blot and histochemical staining. Conclusions The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of pancreas carcinoma throughout progressive developments leading to PDAC. The results would form a firm foundation for future work concerning validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various stages of PDAC. PMID:26262590
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Proteomic approaches in cancer risk and response assessment.
Petricoin, Emanuel F; Liotta, Lance A
2004-02-01
Proteomics is more than just a list-generating exercise where increases or decreases in protein expression are identified. Proteomic technologies will ultimately characterize information-flow through the protein circuitry that interconnects the extracellular microenvironment to the serum or plasma macroenvironment through intracellular signaling systems and their control of gene transcription. The nature of this information can be a cause or a consequence of disease processes and how patients respond to therapy. Analysis of human cancer as a model for how proteomics can have an impact at the bedside can take advantage of several promising new proteomic technologies. These technologies are being developed for early detection and risk assessment, therapeutic targeting and patient-tailored therapy.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Abraham, Paul E.; Garcia, Benjamin J.; Gunter, Lee E.
Drought stress is a recurring feature of world climate and the single most important factor influencing agricultural yield worldwide. Plants display highly variable, species-specific responses to drought and these responses are multifaceted, requiring physiological and morphological changes influenced by genetic and molecular mechanisms. Moreover, the reproducibility of water deficit studies is very cumbersome, which significantly impedes research on drought tolerance, because how a plant responds is highly influenced by the timing, duration, and intensity of the water deficit. Despite progress in the identification of drought-related mechanisms in many plants, the molecular basis of drought resistance remains to be fully understoodmore » in trees, particularly in poplar species because their wide geographic distribution results in varying tolerances to drought. Herein, we aimed to better understand this complex phenomenon in eastern cottonwood ( Populus deltoides) by performing a detailed contrast of the proteome changes between two different water deficit experiments to identify functional intersections and divergences in proteome responses. We investigated plants subjected to cyclic water deficit and compared these responses to plants subjected to prolonged acute water deficit. In total, we identified 108,012 peptide sequences across both experiments that provided insight into the quantitative state of 22,737 Populus gene models and 8,199 functional protein groups in response to drought. Together, these datasets provide the most comprehensive insight into proteome drought responses in poplar to date and a direct proteome comparison between short period dehydration shock and cyclic, post-drought re-watering. Altogether, this investigation provides novel insights into drought avoidance mechanisms that are distinct from progressive drought stress. Additionally, we identified proteins that have been associated as drought-relevant in previous studies. Importantly
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Abraham, Paul E.; Garcia, Benjamin J.; Gunter, Lee E.; ...
2018-02-15
Drought stress is a recurring feature of world climate and the single most important factor influencing agricultural yield worldwide. Plants display highly variable, species-specific responses to drought and these responses are multifaceted, requiring physiological and morphological changes influenced by genetic and molecular mechanisms. Moreover, the reproducibility of water deficit studies is very cumbersome, which significantly impedes research on drought tolerance, because how a plant responds is highly influenced by the timing, duration, and intensity of the water deficit. Despite progress in the identification of drought-related mechanisms in many plants, the molecular basis of drought resistance remains to be fully understoodmore » in trees, particularly in poplar species because their wide geographic distribution results in varying tolerances to drought. Herein, we aimed to better understand this complex phenomenon in eastern cottonwood ( Populus deltoides) by performing a detailed contrast of the proteome changes between two different water deficit experiments to identify functional intersections and divergences in proteome responses. We investigated plants subjected to cyclic water deficit and compared these responses to plants subjected to prolonged acute water deficit. In total, we identified 108,012 peptide sequences across both experiments that provided insight into the quantitative state of 22,737 Populus gene models and 8,199 functional protein groups in response to drought. Together, these datasets provide the most comprehensive insight into proteome drought responses in poplar to date and a direct proteome comparison between short period dehydration shock and cyclic, post-drought re-watering. Altogether, this investigation provides novel insights into drought avoidance mechanisms that are distinct from progressive drought stress. Additionally, we identified proteins that have been associated as drought-relevant in previous studies. Importantly
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Clarke, Raedun L.; Robitaille, Aaron M.; Moon, Randall T.; Keller, Gordon
2015-01-01
Summary The in vitro derivation of hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is complicated by the existence of multiple overlapping embryonic blood cell programs called primitive, erythromyeloid progenitor (EMP), and definitive. As HSCs are only generated during the definitive stage of hematopoiesis, deciphering the regulatory pathways that control the emergence of this program and identifying markers that distinguish it from the other programs are essential. To identify definitive specific pathways and marker sets, we used label-free proteomics to determine the proteome of embryo-derived and mouse embryonic stem cell-derived VE-CADHERIN+CD45− definitive hematopoietic progenitors. With this approach, we identified Stat1 as a marker that distinguishes the definitive erythroid lineage from the primitive- and EMP-derived lineages. Additionally, we provide evidence that the generation of the Stat1+ definitive lineage is dependent on Sox17. These findings establish an approach for monitoring the emergence of definitive hematopoiesis in the PSC differentiation cultures. PMID:26267830
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Arends, Jan; Griego, Marcena; Thomanek, Nikolas; Lindemann, Claudia; Kutscher, Blanka; Meyer, Helmut E; Narberhaus, Franz
2018-04-30
Controlling the cellular abundance and proper function of proteins by proteolysis is a universal process in all living organisms. In Escherichia coli, the ATP-dependent Lon protease is crucial for protein quality control and regulatory processes. To understand how diverse substrates are selected and degraded, unbiased global approaches are needed. We employed a quantitative Super-SILAC mass spectrometry approach and compared the proteomes of a lon mutant and a strain producing the protease to discover Lon-dependent physiological functions. To identify Lon substrates, we took advantage of a Lon trapping variant, which is able to translocate substrates but unable to degrade them. Lon-associated proteins were identified by label-free LC-MS/MS. The combination of both approaches revealed a total of 14 novel Lon substrates. Besides the identification of known pathways affected by Lon, for example the superoxide-stress response, our cumulative data suggests previously unrecognized fundamental functions of Lon in sulfur assimilation, nucleotide biosynthesis, amino acid and central energy metabolism. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
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Standardized protocols for quality control of MRM-based plasma proteomic workflows.
Percy, Andrew J; Chambers, Andrew G; Smith, Derek S; Borchers, Christoph H
2013-01-04
Mass spectrometry (MS)-based proteomics is rapidly emerging as a viable technology for the identification and quantitation of biological samples, such as human plasma--the most complex yet commonly employed biofluid in clinical analyses. The transition from a qualitative to quantitative science is required if proteomics is going to successfully make the transition to a clinically useful technique. MS, however, has been criticized for a lack of reproducibility and interlaboratory transferability. Currently, the MS and plasma proteomics communities lack standardized protocols and reagents to ensure that high-quality quantitative data can be accurately and precisely reproduced by laboratories across the world using different MS technologies. Toward addressing this issue, we have developed standard protocols for multiple reaction monitoring (MRM)-based assays with customized isotopically labeled internal standards for quality control of the sample preparation workflow and the MS platform in quantitative plasma proteomic analyses. The development of reference standards and their application to a single MS platform is discussed herein, along with the results from intralaboratory tests. The tests highlighted the importance of the reference standards in assessing the efficiency and reproducibility of the entire bottom-up proteomic workflow and revealed errors related to the sample preparation and performance quality and deficits of the MS and LC systems. Such evaluations are necessary if MRM-based quantitative plasma proteomics is to be used in verifying and validating putative disease biomarkers across different research laboratories and eventually in clinical laboratories.
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Assay Portal | Office of Cancer Clinical Proteomics Research
The CPTAC Assay Portal serves as a centralized public repository of "fit-for-purpose," multiplexed quantitative mass spectrometry-based proteomic targeted assays. Targeted proteomic assays eliminate issues that are commonly observed using conventional protein detection systems.
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Qi, Da; Zhang, Huaizhong; Fan, Jun; Perkins, Simon; Pisconti, Addolorata; Simpson, Deborah M; Bessant, Conrad; Hubbard, Simon; Jones, Andrew R
2015-09-01
The mzQuantML standard has been developed by the Proteomics Standards Initiative for capturing, archiving and exchanging quantitative proteomic data, derived from mass spectrometry. It is a rich XML-based format, capable of representing data about two-dimensional features from LC-MS data, and peptides, proteins or groups of proteins that have been quantified from multiple samples. In this article we report the development of an open source Java-based library of routines for mzQuantML, called the mzqLibrary, and associated software for visualising data called the mzqViewer. The mzqLibrary contains routines for mapping (peptide) identifications on quantified features, inference of protein (group)-level quantification values from peptide-level values, normalisation and basic statistics for differential expression. These routines can be accessed via the command line, via a Java programming interface access or a basic graphical user interface. The mzqLibrary also contains several file format converters, including import converters (to mzQuantML) from OpenMS, Progenesis LC-MS and MaxQuant, and exporters (from mzQuantML) to other standards or useful formats (mzTab, HTML, csv). The mzqViewer contains in-built routines for viewing the tables of data (about features, peptides or proteins), and connects to the R statistical library for more advanced plotting options. The mzqLibrary and mzqViewer packages are available from https://code.google.com/p/mzq-lib/. © 2015 The Authors. PROTEOMICS Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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PatternLab for proteomics 4.0: A one-stop shop for analyzing shotgun proteomic data
Carvalho, Paulo C; Lima, Diogo B; Leprevost, Felipe V; Santos, Marlon D M; Fischer, Juliana S G; Aquino, Priscila F; Moresco, James J; Yates, John R; Barbosa, Valmir C
2017-01-01
PatternLab for proteomics is an integrated computational environment that unifies several previously published modules for analyzing shotgun proteomic data. PatternLab contains modules for formatting sequence databases, performing peptide spectrum matching, statistically filtering and organizing shotgun proteomic data, extracting quantitative information from label-free and chemically labeled data, performing statistics for differential proteomics, displaying results in a variety of graphical formats, performing similarity-driven studies with de novo sequencing data, analyzing time-course experiments, and helping with the understanding of the biological significance of data in the light of the Gene Ontology. Here we describe PatternLab for proteomics 4.0, which closely knits together all of these modules in a self-contained environment, covering the principal aspects of proteomic data analysis as a freely available and easily installable software package. All updates to PatternLab, as well as all new features added to it, have been tested over the years on millions of mass spectra. PMID:26658470
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Global, quantitative and dynamic mapping of protein subcellular localization.
Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg Hh
2016-06-09
Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology.
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Quantitative Proteomic Profiling of Prostate Cancer Reveals a Role for miR-128 in Prostate Cancer*
Khan, Amjad P.; Poisson, Laila M.; Bhat, Vadiraja B.; Fermin, Damian; Zhao, Rong; Kalyana-Sundaram, Shanker; Michailidis, George; Nesvizhskii, Alexey I.; Omenn, Gilbert S.; Chinnaiyan, Arul M.; Sreekumar, Arun
2010-01-01
Multiple, complex molecular events characterize cancer development and progression. Deciphering the molecular networks that distinguish organ-confined disease from metastatic disease may lead to the identification of biomarkers of cancer invasion and disease aggressiveness. Although alterations in gene expression have been extensively quantified during neoplastic progression, complementary analyses of proteomic changes have been limited. Here we interrogate the proteomic alterations in a cohort of 15 prostate-derived tissues that included five each from adjacent benign prostate, clinically localized prostate cancer, and metastatic disease from distant sites. The experimental strategy couples isobaric tags for relative and absolute quantitation with multidimensional liquid phase peptide fractionation followed by tandem mass spectrometry. Over 1000 proteins were quantified across the specimens and delineated into clinically localized and metastatic prostate cancer-specific signatures. Included in these class-specific profiles were both proteins that were known to be dysregulated during prostate cancer progression and new ones defined by this study. Enrichment analysis of the prostate cancer-specific proteomic signature, to gain insight into the functional consequences of these alterations, revealed involvement of miR-128-a/b regulation during prostate cancer progression. This finding was validated using real time PCR analysis for microRNA transcript levels in an independent set of 15 clinical specimens. miR-128 levels were elevated in benign prostate epithelial cell lines compared with invasive prostate cancer cells. Knockdown of miR-128 induced invasion in benign prostate epithelial cells, whereas its overexpression attenuated invasion in prostate cancer cells. Taken together, our profiles of the proteomic alterations of prostate cancer progression revealed miR-128 as a potentially important negative regulator of prostate cancer cell invasion. PMID:19955085
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Wang, Zhenchao; Yu, Lu; Jin, Linhong; Wang, Wenli; Zhao, Qi; Ran, Longlu; Li, Xiangyang; Chen, Zhuo; Guo, Rong; Wei, Yongtian; Yang, Zhongcheng; Liu, Enlong; Hu, Deyu; Song, Baoan
2017-02-22
Diseases caused by southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) considerably decrease grain yield. Therefore, determining rice cultivars with high resistance to SRBSDV and RRSV is necessary. In this study, rice cultivars with high resistance to SRBSDV and RRSV were evaluated through field trials in Shidian and Mangshi county, Yunnan province, China. SYBR Green I-based quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to quantitatively detect virus gene expression levels in different rice varieties. The following parameters were applied to evaluate rice resistance: acre yield (A.Y.), incidence of infected plants (I.I.P.), virus load (V.L.), disease index (D.I.), and insect quantity (I.Q.) per 100 clusters. Zhongzheyou1 (Z1) and Liangyou2186 (L2186) were considered the most suitable varieties with integrated higher A.Y., lower I.I.P., V.L., D.I. and I.Q. In order to investigate the mechanism of rice resistance, comparative label-free shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) proteomic approaches were applied to comprehensively describe the proteomics of rice varieties' SRBSDV tolerance. Systemic acquired resistance (SAR)-related proteins in Z1 and L2186 may result in the superior resistance of these varieties compared with Fengyouxiangzhan (FYXZ).
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Jarnuczak, Andrew F; Eyers, Claire E; Schwartz, Jean-Marc; Grant, Christopher M; Hubbard, Simon J
2015-09-01
Molecular chaperones play an important role in protein homeostasis and the cellular response to stress. In particular, the HSP70 chaperones in yeast mediate a large volume of protein folding through transient associations with their substrates. This chaperone interaction network can be disturbed by various perturbations, such as environmental stress or a gene deletion. Here, we consider deletions of two major chaperone proteins, SSA1 and SSB1, from the chaperone network in Sacchromyces cerevisiae. We employ a SILAC-based approach to examine changes in global and local protein abundance and rationalise our results via network analysis and graph theoretical approaches. Although the deletions result in an overall increase in intracellular protein content, correlated with an increase in cell size, this is not matched by substantial changes in individual protein concentrations. Despite the phenotypic robustness to deletion of these major hub proteins, it cannot be simply explained by the presence of paralogues. Instead, network analysis and a theoretical consideration of folding workload suggest that the robustness to perturbation is a product of the overall network structure. This highlights how quantitative proteomics and systems modelling can be used to rationalise emergent network properties, and how the HSP70 system can accommodate the loss of major hubs. © 2015 The Authors. PROTEOMICS published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Yu, Haizhong; Wang, Xueyang; Xu, Jiaping; Ma, Yan; Zhang, Shangzhi; Yu, Dong; Fei, Dongqiong; Muhammad, Azharuddin
2017-08-08
Bombyx mori nucleopolyhedrovirus (BmNPV) has been identified as a major pathogen responsible for severe economic loss. Most silkworm strains are susceptible to BmNPV, with only a few highly resistant strains thus far identified. Here we investigated the molecular basis of silkworm resistance to BmNPV using susceptible (the recurrent parent P50) and resistant (near-isogenic line BC9) strains and a combination of iTRAQ-based quantitative proteomics, reverse-transcription quantitative PCR and Western blotting. By comparing the proteomes of infected and non-infected P50 and BC9 silkworms, we identified 793 differentially expressed proteins (DEPs). By gene ontology and KEGG enrichment analyses, we found that these DEPs are preferentially involved in metabolism, catalytic activity, amino sugar and nucleotide sugar metabolism and carbon metabolism. 114 (14.38%) DEPs were associated with the cytoskeleton, immune response, apoptosis, ubiquitination, translation, ion transport, endocytosis and endopeptidase activity. After removing the genetic background and individual immune stress response proteins, we identified 84 DEPs were found that are potentially involved in resistance to BmNPV. Further studies showed that a serine protease was down-regulated in P50 and up-regulated in BC9 after BmNPV infection. Taken together, these results provide insights into the molecular mechanism of silkworm response to BmNPV. Bombyx mori nucleopolyhedrovirus (BmNPV) is highly pathogenic, causing serious losses in sericulture every year. However, the molecular mechanisms of BmNPV infection and host defence remain unclear. Here we combined quantitative proteomic, bioinformatics, RT-qPCR and Western blotting analyses and found that BmNPV invasion causes complex protein alterations in the larval midgut, and that these changes are related to cytoskeleton, immune response, apoptosis, ubiquitination, translation, ion transport, endocytosis and endopeptidase activity. Five important differentially
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Galisson, Frederic; Mahrouche, Louiza; Courcelles, Mathieu; Bonneil, Eric; Meloche, Sylvain; Chelbi-Alix, Mounira K.; Thibault, Pierre
2011-01-01
The small ubiquitin-related modifier (SUMO) is a small group of proteins that are reversibly attached to protein substrates to modify their functions. The large scale identification of protein SUMOylation and their modification sites in mammalian cells represents a significant challenge because of the relatively small number of in vivo substrates and the dynamic nature of this modification. We report here a novel proteomics approach to selectively enrich and identify SUMO conjugates from human cells. We stably expressed different SUMO paralogs in HEK293 cells, each containing a His6 tag and a strategically located tryptic cleavage site at the C terminus to facilitate the recovery and identification of SUMOylated peptides by affinity enrichment and mass spectrometry. Tryptic peptides with short SUMO remnants offer significant advantages in large scale SUMOylome experiments including the generation of paralog-specific fragment ions following CID and ETD activation, and the identification of modified peptides using conventional database search engines such as Mascot. We identified 205 unique protein substrates together with 17 precise SUMOylation sites present in 12 SUMO protein conjugates including three new sites (Lys-380, Lys-400, and Lys-497) on the protein promyelocytic leukemia. Label-free quantitative proteomics analyses on purified nuclear extracts from untreated and arsenic trioxide-treated cells revealed that all identified SUMOylated sites of promyelocytic leukemia were differentially SUMOylated upon stimulation. PMID:21098080
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Gunawardena, Harsha P; O'Brien, Jonathon; Wrobel, John A; Xie, Ling; Davies, Sherri R; Li, Shunqiang; Ellis, Matthew J; Qaqish, Bahjat F; Chen, Xian
2016-02-01
Single quantitative platforms such as label-based or label-free quantitation (LFQ) present compromises in accuracy, precision, protein sequence coverage, and speed of quantifiable proteomic measurements. To maximize the quantitative precision and the number of quantifiable proteins or the quantifiable coverage of tissue proteomes, we have developed a unified approach, termed QuantFusion, that combines the quantitative ratios of all peptides measured by both LFQ and label-based methodologies. Here, we demonstrate the use of QuantFusion in determining the proteins differentially expressed in a pair of patient-derived tumor xenografts (PDXs) representing two major breast cancer (BC) subtypes, basal and luminal. Label-based in-spectra quantitative peptides derived from amino acid-coded tagging (AACT, also known as SILAC) of a non-malignant mammary cell line were uniformly added to each xenograft with a constant predefined ratio, from which Ratio-of-Ratio estimates were obtained for the label-free peptides paired with AACT peptides in each PDX tumor. A mixed model statistical analysis was used to determine global differential protein expression by combining complementary quantifiable peptide ratios measured by LFQ and Ratio-of-Ratios, respectively. With minimum number of replicates required for obtaining the statistically significant ratios, QuantFusion uses the distinct mechanisms to "rescue" the missing data inherent to both LFQ and label-based quantitation. Combined quantifiable peptide data from both quantitative schemes increased the overall number of peptide level measurements and protein level estimates. In our analysis of the PDX tumor proteomes, QuantFusion increased the number of distinct peptide ratios by 65%, representing differentially expressed proteins between the BC subtypes. This quantifiable coverage improvement, in turn, not only increased the number of measurable protein fold-changes by 8% but also increased the average precision of quantitative
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Ichibangase, Tomoko; Sugawara, Yasuhiro; Yamabe, Akio; Koshiyama, Akiyo; Yoshimura, Akari; Enomoto, Takemi; Imai, Kazuhiro
2012-01-01
Systems biology aims to understand biological phenomena in terms of complex biological and molecular interactions, and thus proteomics plays an important role in elucidating protein networks. However, many proteomic methods have suffered from their high variability, resulting in only showing altered protein names. Here, we propose a strategy for elucidating cellular protein networks based on an FD-LC-MS/MS proteomic method. The strategy permits reproducible relative quantitation of differences in protein levels between different cell populations and allows for integration of the data with those obtained through other methods. We demonstrate the validity of the approach through a comparison of differential protein expression in normal and conditional superoxide dismutase 1 gene knockout cells and believe that beginning with an FD-LC-MS/MS proteomic approach will enable researchers to elucidate protein networks more easily and comprehensively. PMID:23029042
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Mi, Yuanyuan; Sun, Chuanyu; Wei, Bingbing; Sun, Feiyu; Guo, Yijun; Hu, Qingfeng; Ding, Weihong; Zhu, Lijie; Xia, Guowei
2018-01-01
Label-free quantitative proteomics has broad applications in the identification of differentially expressed proteins. Here, we applied this method to identify differentially expressed proteins (such as coatomer subunit beta 2 [COPB2]) and evaluated the functions and molecular mechanisms of these proteins in prostate cancer (PCA) cell proliferation. Proteins extracted from surgically resected PCA tissues and adjacent tissues of 3 patients were analyzed by label-free quantitative proteomics. The target protein was confirmed by bioinformatics and GEO dataset analyses. To investigate the role of the target protein in PCA, we used lentivirus-mediated small-interfering RNA (siRNA) to knockdown protein expression in the prostate carcinoma cell line, CWR22RV1 cells and assessed gene and protein expression by reverse transcription quantitative polymerase chain reaction and western blotting. CCK8 and colony formation assays were conducted to evaluate cell proliferation. Cell cycle distributions and apoptosis were assayed by flow cytometry. We selected the differentiation-related protein COPB2 as our target protein based on the results of label-free quantitative proteomics. High expression of COPB2 was found in PCA tissue and was related to poor overall survival based on a public dataset. Cell proliferation was significantly inhibited in COPB2-knockdown CWR22RV1 cells, as demonstrated by CCK8 and colony formation assays. Additionally, the apoptosis rate and percentage of cells in the G 1 phase were increased in COPB2-knockdown cells compared with those in control cells. CDK2, CDK4, and cyclin D1 were downregulated, whereas p21 Waf1/Cip1 and p27 Kip1 were upregulated, affecting the cell cycle signaling pathway. COPB2 significantly promoted CWR22RV1 cell proliferation through the cell cycle signaling pathway. Thus, silencing of COPB2 may have therapeutic applications in PCA. Copyright © 2017 Elsevier Inc. All rights reserved.
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Lancaster, T S; Jefferson, S J; Hunter, J Craig; Lopez, Veronica; Van Eyk, J E; Lakatta, E G; Korzick, D H
2012-10-17
The incidence of myocardial infarction rises sharply at menopause, implicating a potential role for estrogen (E(2)) loss in age-related increases in ischemic injury. We aimed to identify quantitative changes to the cardiac mitochondrial proteome of aging females, based on the hypothesis that E(2) deficiency exacerbates age-dependent disruptions in mitochondrial proteins. Mitochondria isolated from left ventricles of adult (6 mo) and aged (24 mo) F344 ovary-intact or ovariectomized (OVX) rats were labeled with 8plex isobaric tags for relative and absolute quantification (iTRAQ; n = 5-6/group). Groups studied were adult, adult OVX, aged, and aged OVX. In vivo coronary artery ligation and in vitro mitochondrial respiration studies were also performed in a subset of rats. We identified 965 proteins across groups and significant directional changes in 67 proteins of aged and/or aged OVX; 32 proteins were unique to aged OVX. Notably, only six proteins were similarly altered in adult OVX (voltage-dependent ion channel 1, adenine nucleotide translocator 1, cytochrome c oxidase subunits VIIc and VIc, catalase, and myosin binding protein C). Proteins affected by aging were primarily related to cellular metabolism, oxidative stress, and cell death. The largest change occurred in monoamine oxidase-A (MAO-A), a source of oxidative stress. While acute MAO-A inhibition induced mild uncoupling in aged mitochondria, reductions in infarct size were not observed. Age-dependent alterations in mitochondrial signaling indicate a highly selective myocardial response to E(2) deficiency. The combined proteomic and functional approaches described here offer possibility of new protein targets for experimentation and therapeutic intervention in the aged female population.
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Dislich, Bastian; Wohlrab, Felix; Bachhuber, Teresa; Müller, Stephan A.; Kuhn, Peer-Hendrik; Hogl, Sebastian; Meyer-Luehmann, Melanie; Lichtenthaler, Stefan F.
2015-01-01
Analysis of murine cerebrospinal fluid (CSF) by quantitative mass spectrometry is challenging because of low CSF volume, low total protein concentration, and the presence of highly abundant proteins such as albumin. We demonstrate that the CSF proteome of individual mice can be analyzed in a quantitative manner to a depth of several hundred proteins in a robust and simple workflow consisting of single ultra HPLC runs on a benchtop mass spectrometer. The workflow is validated by a comparative analysis of BACE1−/− and wild-type mice using label-free quantification. The protease BACE1 cleaves the amyloid precursor protein (APP) as well as several other substrates and is a major drug target in Alzheimer's disease. We identified a total of 715 proteins with at least 2 unique peptides and quantified 522 of those proteins in CSF from BACE1−/− and wild-type mice. Several proteins, including the known BACE1 substrates APP, APLP1, CHL1 and contactin-2 showed lower abundance in the CSF of BACE1−/− mice, demonstrating that BACE1 substrate identification is possible from CSF. Additionally, ectonucleotide pyrophosphatase 5 was identified as a novel BACE1 substrate and validated in cells using immunoblots and by an in vitro BACE1 protease assay. Likewise, receptor-type tyrosine-protein phosphatase N2 and plexin domain-containing 2 were confirmed as BACE1 substrates by in vitro assays. Taken together, our study shows the deepest characterization of the mouse CSF proteome to date and the first quantitative analysis of the CSF proteome of individual mice. The BACE1 substrates identified in CSF may serve as biomarkers to monitor BACE1 activity in Alzheimer patients treated with BACE inhibitors. PMID:26139848
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Wang, Hongbin; Zhang, Yongqian; Gui, Shuqi; Zhang, Yong; Lu, Fuping; Deng, Yulin
2017-08-15
Comparisons across large numbers of samples are frequently necessary in quantitative proteomics. Many quantitative methods used in proteomics are based on stable isotope labeling, but most of these are only useful for comparing two samples. For up to eight samples, the iTRAQ labeling technique can be used. For greater numbers of samples, the label-free method has been used, but this method was criticized for low reproducibility and accuracy. An ingenious strategy has been introduced, comparing each sample against a 18 O-labeled reference sample that was created by pooling equal amounts of all samples. However, it is necessary to use proportion-known protein mixtures to investigate and evaluate this new strategy. Another problem for comparative proteomics of multiple samples is the poor coincidence and reproducibility in protein identification results across samples. In present study, a method combining 18 O-reference strategy and a quantitation and identification-decoupled strategy was investigated with proportion-known protein mixtures. The results obviously demonstrated that the 18 O-reference strategy had greater accuracy and reliability than other previously used comparison methods based on transferring comparison or label-free strategies. By the decoupling strategy, the quantification data acquired by LC-MS and the identification data acquired by LC-MS/MS are matched and correlated to identify differential expressed proteins, according to retention time and accurate mass. This strategy made protein identification possible for all samples using a single pooled sample, and therefore gave a good reproducibility in protein identification across multiple samples, and allowed for optimizing peptide identification separately so as to identify more proteins. Copyright © 2017 Elsevier B.V. All rights reserved.
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The developmental proteome of Drosophila melanogaster
Casas-Vila, Nuria; Bluhm, Alina; Sayols, Sergi; Dinges, Nadja; Dejung, Mario; Altenhein, Tina; Kappei, Dennis; Altenhein, Benjamin; Roignant, Jean-Yves; Butter, Falk
2017-01-01
Drosophila melanogaster is a widely used genetic model organism in developmental biology. While this model organism has been intensively studied at the RNA level, a comprehensive proteomic study covering the complete life cycle is still missing. Here, we apply label-free quantitative proteomics to explore proteome remodeling across Drosophila’s life cycle, resulting in 7952 proteins, and provide a high temporal-resolved embryogenesis proteome of 5458 proteins. Our proteome data enabled us to monitor isoform-specific expression of 34 genes during development, to identify the pseudogene Cyp9f3Ψ as a protein-coding gene, and to obtain evidence of 268 small proteins. Moreover, the comparison with available transcriptomic data uncovered examples of poor correlation between mRNA and protein, underscoring the importance of proteomics to study developmental progression. Data integration of our embryogenesis proteome with tissue-specific data revealed spatial and temporal information for further functional studies of yet uncharacterized proteins. Overall, our high resolution proteomes provide a powerful resource and can be explored in detail in our interactive web interface. PMID:28381612
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Proteomics wants cRacker: automated standardized data analysis of LC-MS derived proteomic data.
Zauber, Henrik; Schulze, Waltraud X
2012-11-02
The large-scale analysis of thousands of proteins under various experimental conditions or in mutant lines has gained more and more importance in hypothesis-driven scientific research and systems biology in the past years. Quantitative analysis by large scale proteomics using modern mass spectrometry usually results in long lists of peptide ion intensities. The main interest for most researchers, however, is to draw conclusions on the protein level. Postprocessing and combining peptide intensities of a proteomic data set requires expert knowledge, and the often repetitive and standardized manual calculations can be time-consuming. The analysis of complex samples can result in very large data sets (lists with several 1000s to 100,000 entries of different peptides) that cannot easily be analyzed using standard spreadsheet programs. To improve speed and consistency of the data analysis of LC-MS derived proteomic data, we developed cRacker. cRacker is an R-based program for automated downstream proteomic data analysis including data normalization strategies for metabolic labeling and label free quantitation. In addition, cRacker includes basic statistical analysis, such as clustering of data, or ANOVA and t tests for comparison between treatments. Results are presented in editable graphic formats and in list files.
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iTRAQ Quantitative Proteomic Analysis of Vitreous from Patients with Retinal Detachment.
Santos, Fátima Milhano; Gaspar, Leonor Mesquita; Ciordia, Sergio; Rocha, Ana Sílvia; Castro E Sousa, João Paulo; Paradela, Alberto; Passarinha, Luís António; Tomaz, Cândida Teixeira
2018-04-11
Rhegmatogenous retinal detachment (RRD) is a potentially blinding condition characterized by a physical separation between neurosensory retina and retinal pigment epithelium. Quantitative proteomics can help to understand the changes that occur at the cellular level during RRD, providing additional information about the molecular mechanisms underlying its pathogenesis. In the present study, iTRAQ labeling was combined with two-dimensional LC-ESI-MS/MS to find expression changes in the proteome of vitreous from patients with RRD when compared to control samples. A total of 150 proteins were found differentially expressed in the vitreous of patients with RRD, including 96 overexpressed and 54 underexpressed. Several overexpressed proteins, several such as glycolytic enzymes (fructose-bisphosphate aldolase A, gamma-enolase, and phosphoglycerate kinase 1), glucose transporters (GLUT-1), growth factors (metalloproteinase inhibitor 1), and serine protease inhibitors (plasminogen activator inhibitor 1) are regulated by HIF-1, which suggests that HIF-1 signaling pathway can be triggered in response to RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Nevertheless, the differentially expressed proteins found in this study suggest that different mechanisms are activated after RRD to promote the survival of retinal cells through complex cellular responses.
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iTRAQ Quantitative Proteomic Analysis of Vitreous from Patients with Retinal Detachment
Gaspar, Leonor Mesquita; Ciordia, Sergio; Rocha, Ana Sílvia; Castro e Sousa, João Paulo; Paradela, Alberto
2018-01-01
Rhegmatogenous retinal detachment (RRD) is a potentially blinding condition characterized by a physical separation between neurosensory retina and retinal pigment epithelium. Quantitative proteomics can help to understand the changes that occur at the cellular level during RRD, providing additional information about the molecular mechanisms underlying its pathogenesis. In the present study, iTRAQ labeling was combined with two-dimensional LC-ESI-MS/MS to find expression changes in the proteome of vitreous from patients with RRD when compared to control samples. A total of 150 proteins were found differentially expressed in the vitreous of patients with RRD, including 96 overexpressed and 54 underexpressed. Several overexpressed proteins, several such as glycolytic enzymes (fructose-bisphosphate aldolase A, gamma-enolase, and phosphoglycerate kinase 1), glucose transporters (GLUT-1), growth factors (metalloproteinase inhibitor 1), and serine protease inhibitors (plasminogen activator inhibitor 1) are regulated by HIF-1, which suggests that HIF-1 signaling pathway can be triggered in response to RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Nevertheless, the differentially expressed proteins found in this study suggest that different mechanisms are activated after RRD to promote the survival of retinal cells through complex cellular responses. PMID:29641463
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Koczorowska, Maria Magdalena; Friedemann, Charlotte; Geiger, Klaus; Follo, Marie; Biniossek, Martin Lothar; Schilling, Oliver
2017-12-01
Solid tumors contain various components that together form the tumor microenvironment. Cancer associated fibroblasts (CAFs) are capable of secreting and responding to signaling molecules and growth factors. Due to their role in tumor development, CAFs are considered as potential therapeutic targets. A prominent tumor-associated signaling molecule is transforming growth factor β (TGFβ), an inducer of epithelial-to-mesenchymal transition (EMT). The differential action of TGFβ on CAFs and ETCs (epithelial tumor cells) has recently gained interest. Here, we aimed to investigate the effects of TGFβ on CAFs and ETCs at the proteomic level. We established a 2D co-culture system of differentially fluorescently labeled CAFs and ETCs and stimulated this co-culture system with TGFβ. The respective cell types were separated using FACS and subjected to quantitative analyses of individual proteomes using mass spectrometry. We found that TGFβ treatment had a strong impact on the proteome composition of CAFs, whereas ETCs responded only marginally to TGFβ. Quantitative proteomic analyses of the different cell types revealed up-regulation of extracellular matrix (ECM) proteins in TGFβ treated CAFs. In addition, we found that the TGFβ treated CAFs exhibited increased N-cadherin levels. From our data we conclude that CAFs respond to TGFβ treatment by changing their proteome composition, while ETCs appear to be rather resilient.
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Cell death proteomics database: consolidating proteomics data on cell death.
Arntzen, Magnus Ø; Bull, Vibeke H; Thiede, Bernd
2013-05-03
Programmed cell death is a ubiquitous process of utmost importance for the development and maintenance of multicellular organisms. More than 10 different types of programmed cell death forms have been discovered. Several proteomics analyses have been performed to gain insight in proteins involved in the different forms of programmed cell death. To consolidate these studies, we have developed the cell death proteomics (CDP) database, which comprehends data from apoptosis, autophagy, cytotoxic granule-mediated cell death, excitotoxicity, mitotic catastrophe, paraptosis, pyroptosis, and Wallerian degeneration. The CDP database is available as a web-based database to compare protein identifications and quantitative information across different experimental setups. The proteomics data of 73 publications were integrated and unified with protein annotations from UniProt-KB and gene ontology (GO). Currently, more than 6,500 records of more than 3,700 proteins are included in the CDP. Comparing apoptosis and autophagy using overrepresentation analysis of GO terms, the majority of enriched processes were found in both, but also some clear differences were perceived. Furthermore, the analysis revealed differences and similarities of the proteome between autophagosomal and overall autophagy. The CDP database represents a useful tool to consolidate data from proteome analyses of programmed cell death and is available at http://celldeathproteomics.uio.no.
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Huang, Rongrong; Chen, Zhongsi; He, Lei; He, Nongyue; Xi, Zhijiang; Li, Zhiyang; Deng, Yan; Zeng, Xin
2017-01-01
There is a critical need for the discovery of novel biomarkers for early detection and targeted therapy of cancer, a major cause of deaths worldwide. In this respect, proteomic technologies, such as mass spectrometry (MS), enable the identification of pathologically significant proteins in various types of samples. MS is capable of high-throughput profiling of complex biological samples including blood, tissues, urine, milk, and cells. MS-assisted proteomics has contributed to the development of cancer biomarkers that may form the foundation for new clinical tests. It can also aid in elucidating the molecular mechanisms underlying cancer. In this review, we discuss MS principles and instrumentation as well as approaches in MS-based proteomics, which have been employed in the development of potential biomarkers. Furthermore, the challenges in validation of MS biomarkers for their use in clinical practice are also reviewed. PMID:28912895
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RECENT ADVANCES IN QUANTITATIVE NEUROPROTEOMICS
Craft, George E; Chen, Anshu; Nairn, Angus C
2014-01-01
The field of proteomics is undergoing rapid development in a number of different areas including improvements in mass spectrometric platforms, peptide identification algorithms and bioinformatics. In particular, new and/or improved approaches have established robust methods that not only allow for in-depth and accurate peptide and protein identification and modification, but also allow for sensitive measurement of relative or absolute quantitation. These methods are beginning to be applied to the area of neuroproteomics, but the central nervous system poses many specific challenges in terms of quantitative proteomics, given the large number of different neuronal cell types that are intermixed and that exhibit distinct patterns of gene and protein expression. This review highlights the recent advances that have been made in quantitative neuroproteomics, with a focus on work published over the last five years that applies emerging methods to normal brain function as well as to various neuropsychiatric disorders including schizophrenia and drug addiction as well as of neurodegenerative diseases including Parkinson’s disease and Alzheimer’s disease. While older methods such as two-dimensional polyacrylamide electrophoresis continued to be used, a variety of more in-depth MS-based approaches including both label (ICAT, iTRAQ, TMT, SILAC, SILAM), label-free (label-free, MRM, SWATH) and absolute quantification methods, are rapidly being applied to neurobiological investigations of normal and diseased brain tissue as well as of cerebrospinal fluid (CSF). While the biological implications of many of these studies remain to be clearly established, that there is a clear need for standardization of experimental design and data analysis, and that the analysis of protein changes in specific neuronal cell types in the central nervous system remains a serious challenge, it appears that the quality and depth of the more recent quantitative proteomics studies is beginning to
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Recent advances in quantitative neuroproteomics.
Craft, George E; Chen, Anshu; Nairn, Angus C
2013-06-15
The field of proteomics is undergoing rapid development in a number of different areas including improvements in mass spectrometric platforms, peptide identification algorithms and bioinformatics. In particular, new and/or improved approaches have established robust methods that not only allow for in-depth and accurate peptide and protein identification and modification, but also allow for sensitive measurement of relative or absolute quantitation. These methods are beginning to be applied to the area of neuroproteomics, but the central nervous system poses many specific challenges in terms of quantitative proteomics, given the large number of different neuronal cell types that are intermixed and that exhibit distinct patterns of gene and protein expression. This review highlights the recent advances that have been made in quantitative neuroproteomics, with a focus on work published over the last five years that applies emerging methods to normal brain function as well as to various neuropsychiatric disorders including schizophrenia and drug addiction as well as of neurodegenerative diseases including Parkinson's disease and Alzheimer's disease. While older methods such as two-dimensional polyacrylamide electrophoresis continued to be used, a variety of more in-depth MS-based approaches including both label (ICAT, iTRAQ, TMT, SILAC, SILAM), label-free (label-free, MRM, SWATH) and absolute quantification methods, are rapidly being applied to neurobiological investigations of normal and diseased brain tissue as well as of cerebrospinal fluid (CSF). While the biological implications of many of these studies remain to be clearly established, that there is a clear need for standardization of experimental design and data analysis, and that the analysis of protein changes in specific neuronal cell types in the central nervous system remains a serious challenge, it appears that the quality and depth of the more recent quantitative proteomics studies is beginning to shed
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Li, Cheng; Zhang, Yong; Xie, Zhang-Xian; He, Zhi-Ping; Lin, Lin; Wang, Da-Zhi
2013-06-28
contentious and the evolutionary relationships within the complex are unclear, which has seriously impeded our understanding of Alexandrium-causing HABs and, consequently, the monitoring, mitigation and prevention. Technical significance: This study, for the first time, compared the global protein expression patterns of eight ecotypes from the A. tamarense complex and identified species-specific peptides using a quantitative proteomic approach combining 2-D DIGE and MALDI-TOF/TOF MS. This study demonstrated that the evolutionary divergence had occurred in the A. tamarense complex at the proteomic level, and the complex should be classified into three species, i.e. A. tamarense, A. catenella, and A. fundyense. Moreover, the species-specific peptide mass fingerprints could be used as potential biomarkers to distinguish the three morphotypes. Copyright © 2013 Elsevier B.V. All rights reserved.
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Shao, Shiying; Guo, Tiannan; Gross, Vera; Lazarev, Alexander; Koh, Ching Chiek; Gillessen, Silke; Joerger, Markus; Jochum, Wolfram; Aebersold, Ruedi
2016-06-03
The reproducible and efficient extraction of proteins from biopsy samples for quantitative analysis is a critical step in biomarker and translational research. Recently, we described a method consisting of pressure-cycling technology (PCT) and sequential windowed acquisition of all theoretical fragment ions-mass spectrometry (SWATH-MS) for the rapid quantification of thousands of proteins from biopsy-size tissue samples. As an improvement of the method, we have incorporated the PCT-MicroPestle into the PCT-SWATH workflow. The PCT-MicroPestle is a novel, miniaturized, disposable mechanical tissue homogenizer that fits directly into the microTube sample container. We optimized the pressure-cycling conditions for tissue lysis with the PCT-MicroPestle and benchmarked the performance of the system against the conventional PCT-MicroCap method using mouse liver, heart, brain, and human kidney tissues as test samples. The data indicate that the digestion of the PCT-MicroPestle-extracted proteins yielded 20-40% more MS-ready peptide mass from all tissues tested with a comparable reproducibility when compared to the conventional PCT method. Subsequent SWATH-MS analysis identified a higher number of biologically informative proteins from a given sample. In conclusion, we have developed a new device that can be seamlessly integrated into the PCT-SWATH workflow, leading to increased sample throughput and improved reproducibility at both the protein extraction and proteomic analysis levels when applied to the quantitative proteomic analysis of biopsy-level samples.
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Han, Kaikai; Zhao, Dongmin; Liu, Yuzhuo; Liu, Qingtao; Huang, Xinmei; Yang, Jing; An, Fengjiao; Li, Yin
2016-01-01
Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. DTMUV infection mainly results in significant decreases in egg production in egg-laying ducks within 1–2 weeks post infection. However, information on the comparative protein expression of host tissues in response to DTMUV infection is limited. In the present study, the cellular protein response to DTMUV infection in duck ovarian follicles was analyzed using nano-flow high-performance liquid chromatography-electrospray tandem mass spectrometry. Quantitative proteomic analysis revealed 131 differentially expressed proteins, among which 53 were up regulated and 78 were down regulated. The identified proteins were involved in the regulation of essential processes such as cellular structure and integrity, RNA processing, protein biosynthesis and modification, vesicle transport, signal transduction, and mitochondrial pathway. Some selected proteins that were found to be regulated in DTMUV-infected tissues were screened by quantitative real-time PCR to examine their regulation at the transcriptional level, western blot analysis was used to validate the changes of some selected proteins on translational level. To our knowledge, this study is the first to analyze the proteomic changes in duck ovarian follicles following DTMUV infection. The protein-related information obtained in this study may be useful to understand the host response to DTMUV infection and the inherent mechanism of DTMUV replication and pathogenicity. PMID:27066001
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ITRAQ-based quantitative proteomic analysis of Cynops orientalis limb regeneration.
Tang, Jie; Yu, Yuan; Zheng, Hanxue; Yin, Lu; Sun, Mei; Wang, Wenjun; Cui, Jihong; Liu, Wenguang; Xie, Xin; Chen, Fulin
2017-09-22
Salamanders regenerate their limbs after amputation. However, the molecular mechanism of this unique regeneration remains unclear. In this study, isobaric tags for relative and absolute quantification (iTRAQ) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) was employed to quantitatively identify differentially expressed proteins in regenerating limbs 3, 7, 14, 30 and 42 days post amputation (dpa). Of 2636 proteins detected in total, 253 proteins were differentially expressed during different regeneration stages. Among these proteins, Asporin, Cadherin-13, Keratin, Collagen alpha-1(XI) and Titin were down-regulated. CAPG, Coronin-1A, AnnexinA1, Cathepsin B were up-regulated compared with the control. The identified proteins were further analyzed to obtain information about their expression patterns and functions in limb regeneration. Functional analysis indicated that the differentially expressed proteins were associated with wound healing, immune response, cellular process, metabolism and binding. This work indicated that significant proteome alternations occurred during salamander limb regeneration. The results may provide fundamental knowledge to understand the mechanism of limb regeneration.
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Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes in salt tolerance.
Barkla, Bronwyn J; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar
2009-12-01
To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na(+) sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H(+)-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H(+)-pump activity.
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Friedman, David B
2012-01-01
All quantitative proteomics experiments measure variation between samples. When performing large-scale experiments that involve multiple conditions or treatments, the experimental design should include the appropriate number of individual biological replicates from each condition to enable the distinction between a relevant biological signal from technical noise. Multivariate statistical analyses, such as principal component analysis (PCA), provide a global perspective on experimental variation, thereby enabling the assessment of whether the variation describes the expected biological signal or the unanticipated technical/biological noise inherent in the system. Examples will be shown from high-resolution multivariable DIGE experiments where PCA was instrumental in demonstrating biologically significant variation as well as sample outliers, fouled samples, and overriding technical variation that would not be readily observed using standard univariate tests.
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Global, quantitative and dynamic mapping of protein subcellular localization
Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg HH
2016-01-01
Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology. DOI: http://dx.doi.org/10.7554/eLife.16950.001 PMID:27278775
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Proteomics: a new approach to the study of disease.
Chambers, G; Lawrie, L; Cash, P; Murray, G I
2000-11-01
The global analysis of cellular proteins has recently been termed proteomics and is a key area of research that is developing in the post-genome era. Proteomics uses a combination of sophisticated techniques including two-dimensional (2D) gel electrophoresis, image analysis, mass spectrometry, amino acid sequencing, and bio-informatics to resolve comprehensively, to quantify, and to characterize proteins. The application of proteomics provides major opportunities to elucidate disease mechanisms and to identify new diagnostic markers and therapeutic targets. This review aims to explain briefly the background to proteomics and then to outline proteomic techniques. Applications to the study of human disease conditions ranging from cancer to infectious diseases are reviewed. Finally, possible future advances are briefly considered, especially those which may lead to faster sample throughput and increased sensitivity for the detection of individual proteins. Copyright 2000 John Wiley & Sons, Ltd.
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Sethi, Manveen K; Thaysen-Andersen, Morten; Kim, Hoguen; Park, Cheol Keun; Baker, Mark S; Packer, Nicolle H; Paik, Young-Ki; Hancock, William S; Fanayan, Susan
2015-08-03
Modern proteomics has proven instrumental in our understanding of the molecular deregulations associated with the development and progression of cancer. Herein, we profile membrane-enriched proteome of tumor and adjacent normal tissues from eight CRC patients using label-free nanoLC-MS/MS-based quantitative proteomics and advanced pathway analysis. Of the 948 identified proteins, 184 proteins were differentially expressed (P<0.05, fold change>1.5) between the tumor and non-tumor tissue (69 up-regulated and 115 down-regulated in tumor tissues). The CRC tumor and non-tumor tissues clustered tightly in separate groups using hierarchical cluster analysis of the differentially expressed proteins, indicating a strong CRC-association of this proteome subset. Specifically, cancer associated proteins such as FN1, TNC, DEFA1, ITGB2, MLEC, CDH17, EZR and pathways including actin cytoskeleton and RhoGDI signaling were deregulated. Stage-specific proteome signatures were identified including up-regulated ribosomal proteins and down-regulated annexin proteins in early stage CRC. Finally, EGFR(+) CRC tissues showed an EGFR-dependent down-regulation of cell adhesion molecules, relative to EGFR(-) tissues. Taken together, this study provides a detailed map of the altered proteome and associated protein pathways in CRC, which enhances our mechanistic understanding of CRC biology and opens avenues for a knowledge-driven search for candidate CRC protein markers. Copyright © 2015 Elsevier B.V. All rights reserved.
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Lata, Manju; Sharma, Divakar; Kumar, Bhavnesh; Deo, Nirmala; Tiwari, Pramod Kumar; Bisht, Deepa; Venkatesan, Krishnamurthy
2015-01-01
Ofloxacin (OFX) and moxifloxacin (MOX) are the most promising second line drugs for tuberculosis treatment. Although the primary mechanism of action of OFX and MOX is gyrase inhibition, other possible mechanisms cannot be ruled out. Being the functional moiety of cell, the proteins act as primary targets for developing drugs, diagnostics and therapeutics. In this study we have investigated the proteomic changes of Mycobacterium tuberculosis isolates induced by OFX and MOX by applying comparative proteomic approaches based on two-dinensional gel electrophoresis (2DE) along with matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF/TOF-MS) and bioinformatic tools. The findings are likely to provide new understanding of OFX and MOX mechanisms that might be helpful in exploring new diagnostics and drug targets. Our study explored eleven proteins (Rv2889c, Rv2623, Rv0952, Rv1827, Rv1932, Rv0054, Rv1080c, Rv3418c, Rv3914, Rv1636 and Rv0009) that were overexpressed in the presence of drugs. Among them, Rv2623, Rv1827 and Rv1636 were identified as proteins with unknown function. InterProScan and molecular docking revealed that the conserved domain of hypothetical proteins interact with OFX and MOX which indicate a probable inhibition/modulation of the functioning of these proteins by both drugs, which might be overexpressed to overcome this effect.
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Hepatic SILAC proteomic data from PANDER transgenic model.
Athanason, Mark G; Stevens, Stanley M; Burkhardt, Brant R
2016-12-01
This article contains raw and processed data related to research published in "Quantitative Proteomic Profiling Reveals Hepatic Lipogenesis and Liver X Receptor Activation in the PANDER Transgenic Model" (M.G. Athanason, W.A. Ratliff, D. Chaput, C.B. MarElia, M.N. Kuehl, S.M., Jr. Stevens, B.R. Burkhardt (2016)) [1], and was generated by "spike-in" SILAC-based proteomic analysis of livers obtained from the PANcreatic-Derived factor (PANDER) transgenic mouse (PANTG) under various metabolic conditions [1]. The mass spectrometry output of the PANTG and wild-type B6SJLF mice liver tissue and resulting proteome search from MaxQuant 1.2.2.5 employing the Andromeda search algorithm against the UniprotKB reference database for Mus musculus has been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository with dataset identifiers PRIDE: PXD004171 and doi:10.6019/PXD004171. Protein ratio values representing PANTG/wild-type obtained by MaxQuant analysis were input into the Perseus processing suite to determine statistical significance using the Significance A outlier test (p<0.05). Differentially expressed proteins using this approach were input into Ingenuity Pathway Analysis to determined altered pathways and upstream regulators that were altered in PANTG mice.
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Quantitative Proteomic Profiling of Low Dose Ionizing Radiation Effects in a Human Skin Model
DOE Office of Scientific and Technical Information (OSTI.GOV)
Hengel, Shawna; Aldrich, Joshua T.; Waters, Katrina M.
2014-07-29
To assess molecular responses to low doses of radiation that may be encountered during medical diagnostic procedures, nuclear accidents, or terrorist acts, a quantitative global proteomic approach was used to identify protein alterations in a reconstituted human skin tissue treated with 10 cGy of ionizing radiation. Subcellular fractionation was employed to remove highly abundant structural proteins and provide insight on radiation induced alterations in protein abundance and localization. In addition, peptides were post-fractionated using high resolution 2-dimensional liquid chromatography to increase the dynamic range of detection of protein abundance and translocation changes. Quantitative data was obtained by labeling peptides withmore » 8-plex isobaric iTRAQ tags. A total of 207 proteins were detected with statistically significant alterations in abundance and/or subcellular localization compared to sham irradiated tissues. Bioinformatics analysis of the data indicated that the top canonical pathways affected by low dose radiation are related to cellular metabolism. Among the proteins showing alterations in abundance, localization and proteolytic processing was the skin barrier protein filaggrin which is consistent with our previous observation that ionizing radiation alters profilaggrin processing with potential effects on skin barrier functions. In addition, a large number of proteases and protease regulators were affected by low dose radiation exposure indicating that altered proteolytic activity may be a hallmark of low dose radiation exposure. While several studies have demonstrated altered transcriptional regulation occurs following low dose radiation exposures, the data presented here indicates post-transcriptional regulation of protein abundance, localization, and proteolytic processing play an important role in regulating radiation responses in complex human tissues.« less
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Development of proteome-wide binding reagents for research and diagnostics.
Taussig, Michael J; Schmidt, Ronny; Cook, Elizabeth A; Stoevesandt, Oda
2013-12-01
Alongside MS, antibodies and other specific protein-binding molecules have a special place in proteomics as affinity reagents in a toolbox of applications for determining protein location, quantitative distribution and function (affinity proteomics). The realisation that the range of research antibodies available, while apparently vast is nevertheless still very incomplete and frequently of uncertain quality, has stimulated projects with an objective of raising comprehensive, proteome-wide sets of protein binders. With progress in automation and throughput, a remarkable number of recent publications refer to the practical possibility of selecting binders to every protein encoded in the genome. Here we review the requirements of a pipeline of production of protein binders for the human proteome, including target prioritisation, antigen design, 'next generation' methods, databases and the approaches taken by ongoing projects in Europe and the USA. While the task of generating affinity reagents for all human proteins is complex and demanding, the benefits of well-characterised and quality-controlled pan-proteome binder resources for biomedical research, industry and life sciences in general would be enormous and justify the effort. Given the technical, personnel and financial resources needed to fulfil this aim, expansion of current efforts may best be addressed through large-scale international collaboration. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Zhang, Yao; Li, Yanchang; Zhang, Yongguang; Wang, Zhiqiang; Zhao, Mingzhi; Su, Na; Zhang, Tao; Chen, Lingsheng; Wei, Wei; Luo, Jing; Zhou, Yanxia; Xu, Yongru; Xu, Ping; Li, Wenjun; Tao, Yong
2016-01-04
The genus Nocardiopsis is one of the most dominant Actinobacteria that survives in hypersaline environments. However, the adaptation mechanisms for halophilism are still unclear. Here, we performed isobaric tags for relative and absolute quantification based quantitative proteomics to investigate the functions of the membrane proteome after salt stress. A total of 683 membrane proteins were identified and quantified, of which 126 membrane proteins displayed salt-induced changes in abundance. Intriguingly, bioinformatics analyses indicated that these differential proteins showed two expression patterns, which were further validated by phenotypic changes and functional differences. The majority of ABC transporters, secondary active transporters, cell motility proteins, and signal transduction kinases were up-regulated with increasing salt concentration, whereas cell differentiation, small molecular transporter (ions and amino acids), and secondary metabolism proteins were significantly up-regulated at optimum salinity, but down-regulated or unchanged at higher salinity. The small molecule transporters and cell differentiation-related proteins acted as sensing proteins that played a more important biological role at optimum salinity. However, the ABC transporters for compatible solutes, Na(+)-dependent transporters, and cell motility proteins acted as adaptive proteins that actively counteracted higher salinity stress. Overall, regulation of membrane proteins may provide a major protection strategy against hyperosmotic stress.
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Top-Down Proteomics and Farm Animal and Aquatic Sciences.
Campos, Alexandre M O; de Almeida, André M
2016-12-21
Proteomics is a field of growing importance in animal and aquatic sciences. Similar to other proteomic approaches, top-down proteomics is slowly making its way within the vast array of proteomic approaches that researchers have access to. This opinion and mini-review article is dedicated to top-down proteomics and how its use can be of importance to animal and aquatic sciences. Herein, we include an overview of the principles of top-down proteomics and how it differs regarding other more commonly used proteomic methods, especially bottom-up proteomics. In addition, we provide relevant sections on how the approach was or can be used as a research tool and conclude with our opinions of future use in animal and aquatic sciences.
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A redox proteomics approach to investigate the mode of action of nanomaterials
DOE Office of Scientific and Technical Information (OSTI.GOV)
Riebeling, Christian; Wiemann, Martin; Schnekenburger, Jürgen
2016-05-15
Numbers of engineered nanomaterials (ENMs) are steadily increasing. Therefore, alternative testing approaches with reduced costs and high predictivity suitable for high throughput screening and prioritization are urgently needed to ensure a fast and effective development of safe products. In parallel, extensive research efforts are targeted to understanding modes of action of ENMs, which may also support the development of new predictive assays. Oxidative stress is a widely accepted paradigm associated with different adverse outcomes of ENMs. It has frequently been identified in in vitro and in vivo studies and different assays have been developed for this purpose. Fluorescent dye basedmore » read-outs are most frequently used for cell testing in vitro but may be limited due to possible interference of the ENMs. Recently, other assays have been put forward such as acellular determination of ROS production potential using methods like electron spin resonance, antioxidant quantification or the use of specific sensors. In addition, Omics based approaches have gained increasing attention. In particular, redox proteomics can combine the assessment of oxidative stress with the advantage of getting more detailed mechanistic information. Here we propose a comprehensive testing strategy for assessing the oxidative stress potential of ENMs, which combines acellular methods and fast in vitro screening approaches, as well as a more involved detailed redox proteomics approach. This allows for screening and prioritization in a first tier and, if required, also for unraveling mechanistic details down to compromised signaling pathways. - Highlights: • Oxidative stress is a general paradigm for nanomaterial hazard mechanism of action. • Reactive oxygen species generation can be predicted using acellular assays. • Cellular assays based on fluorescence suffer from interference by nanomaterials. • Protein carbonylation is an irreversible and predictive mark of oxidative
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Recent advances in proteomic applications for schistosomiasis research: potential clinical impact.
Sotillo, Javier; Doolan, Denise; Loukas, Alex
2017-02-01
Schistosomiasis is a neglected tropical disease affecting hundreds of millions of people worldwide. Recent advances in the field of proteomics and the development of new and highly sensitive mass spectrometers and quantitative techniques have provided new tools for advancing the molecular biology, cell biology, diagnosis and vaccine development for public health threats such as schistosomiasis. Areas covered: In this review we describe the latest advances in research that utilizes proteomics-based tools to address some of the key challenges to developing effective interventions against schistosomiasis. We also provide information about the potential of extracellular vesicles to advance the fight against this devastating disease. Expert commentary: Different proteins are already being tested as vaccines against schistosomiasis with promising results. The re-analysis of the Schistosoma spp. proteomes using new and more sensitive mass spectrometers as well as better separation approaches will help identify more vaccine targets in a rational and informed manner. In addition, the recent development of new proteome microarrays will facilitate characterisation of novel markers of infection as well as new vaccine and diagnostic candidate antigens.
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Lan, Yu-Long; Zhou, Jun-Jun; Liu, Jing; Huo, Xiao-Kui; Wang, Ya-Li; Liang, Jia-Hao; Zhao, Jian-Chao; Sun, Cheng-Peng; Yu, Zhen-Long; Fang, Lin-Lin; Tian, Xiang-Ge; Feng, Lei; Ning, Jing; Zhang, Bao-Jing; Wang, Chao; Zhao, Xin-Yu; Ma, Xiao-Chi
2018-06-21
Uncaria rhynchophylla, known as "Gou-teng", is a traditional Chinese medicine (TCM) used to extinguish wind, clear heat, arrest convulsions, and pacify the liver. Although U. rhynchophylla has a long history of being often used to treat central nervous system (CNS) diseases, its efficacy and potential mechanism are still uncertain. This study investigated neuroprotective effect and the underlying mechanism of U. rhynchophylla extract (URE) in MPP+-induced SH-SY5Y cells and MPTP-induced mice. MPP+-induced SH-SY5Y cells and MPTP-induced mice were used to established Parkinson's disease (PD) models. Quantitative proteomics and bioinformatics were used to uncover proteomics changes of URE. Western blotting was used to validate main differentially expressed proteins and test HSP90 client proteins (apoptosis-related, autophagy-related, MAPKs, PI3K, and AKT proteins). Flow cytometry and JC-1 staining assay were further used to confirm the effect of URE on MPP+-induced apoptosis in SH-SY5Y cells. Gait analysis was used to detect the behavioral changes in MPTP-induced mice. The levels of dopamine (DA) and their metabolites were examined in striatum (STR) by HPLC-EC. The positive expression of tyrosine hydroxylase (TH) was detected by immunohischemical staining and Western blotting. URE dose-dependently increased the cell viability in MPP+-induced SH-SY5Y cells. Quantitative proteomics and bioinformatics results confirmed that HSP90 was an important differentially expressed protein of URE. URE inhibited the expression of HSP90, which further reversed MPP+-induced cell apoptosis and autophagy by increasing the expressions of Bcl-2, Cyclin D1, p-ERK, p-PI3K p85, PI3K p110α, p-AKT, and LC3-I and decreasing cleaved caspase 3, Bax, p-JNK, p-p38, and LC3-II. URE also markedly decreased the apoptotic ratio and elevated mitochondrial transmembrane potential (DΨm). Furthermore, URE treatment ameliorated behavioral impairments, increased the contents of DA and its metabolites and
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Xia, Qiangwei; Wang, Tiansong; Park, Yoonsuk; Lamont, Richard J.; Hackett, Murray
2009-01-01
Differential analysis of whole cell proteomes by mass spectrometry has largely been applied using various forms of stable isotope labeling. While metabolic stable isotope labeling has been the method of choice, it is often not possible to apply such an approach. Four different label free ways of calculating expression ratios in a classic “two-state” experiment are compared: signal intensity at the peptide level, signal intensity at the protein level, spectral counting at the peptide level, and spectral counting at the protein level. The quantitative data were mined from a dataset of 1245 qualitatively identified proteins, about 56% of the protein encoding open reading frames from Porphyromonas gingivalis, a Gram-negative intracellular pathogen being studied under extracellular and intracellular conditions. Two different control populations were compared against P. gingivalis internalized within a model human target cell line. The q-value statistic, a measure of false discovery rate previously applied to transcription microarrays, was applied to proteomics data. For spectral counting, the most logically consistent estimate of random error came from applying the locally weighted scatter plot smoothing procedure (LOWESS) to the most extreme ratios generated from a control technical replicate, thus setting upper and lower bounds for the region of experimentally observed random error. PMID:19337574
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Jiang, Shuai; Qiu, Limei; Wang, Lingling; Jia, Zhihao; Lv, Zhao; Wang, Mengqiang; Liu, Conghui; Xu, Jiachao; Song, Linsheng
2018-01-01
As invertebrates lack an adaptive immune system, they depend to a large extent on their innate immune system to recognize and clear invading pathogens. Although phagocytes play pivotal roles in invertebrate innate immunity, the molecular mechanisms underlying this killing remain unclear. Cells of this type from the Pacific oyster Crassostrea gigas were classified efficiently in this study via fluorescence-activated cell sorting (FACS) based on their phagocytosis of FITC-labeled latex beads. Transcriptomic and quantitative proteomic analyses revealed a series of differentially expressed genes (DEGs) and proteins present in phagocytes; of the 352 significantly high expressed proteins identified here within the phagocyte proteome, 262 corresponding genes were similarly high expressed in the transcriptome, while 140 of 205 significantly low expressed proteins within the proteome were transcriptionally low expressed. A pathway crosstalk network analysis of these significantly high expressed proteins revealed that phagocytes were highly activated in a number of antimicrobial-related biological processes, including oxidation–reduction and lysosomal proteolysis processes. A number of DEGs, including oxidase, lysosomal protease, and immune receptors, were also validated in this study using quantitative PCR, while seven lysosomal cysteine proteases, referred to as cathepsin Ls, were significantly high expressed in phagocytes. Results show that the expression level of cathepsin L protein in phagocytes [mean fluorescence intensity (MFI): 327 ± 51] was significantly higher (p < 0.01) than that in non-phagocytic hemocytes (MFI: 83 ± 26), while the cathepsin L protein was colocalized with the phagocytosed Vibrio splendidus in oyster hemocytes during this process. The results of this study collectively suggest that oyster phagocytes possess both potent oxidative killing and microbial disintegration capacities; these findings provide important insights into hemocyte
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Du, Zhe; Chen, Yinguang; Li, Xu
2017-10-15
Microbial degradation of estrogenic compounds can be affected by the nitrogen source and background carbon in the environment. However, the underlying mechanisms are not well understood. The objective of this study was to elucidate the molecular mechanisms of estrone (E1) biodegradation at the protein level under various background nitrogen (nitrate or ammonium) and carbon conditions (no background carbon, acetic acid, or humic acid as background carbon) by a newly isolated bacterial strain. The E1 degrading bacterial strain, Hydrogenophaga atypica ZD1, was isolated from river sediments and its proteome was characterized under various experimental conditions using quantitative proteomics. Results show that the E1 degradation rate was faster when ammonium was used as the nitrogen source than with nitrate. The degradation rate was also faster when either acetic acid or humic acid was present in the background. Proteomics analyses suggested that the E1 biodegradation products enter the tyrosine metabolism pathway. Compared to nitrate, ammonium likely promoted E1 degradation by increasing the activities of the branched-chain-amino-acid aminotransferase (IlvE) and enzymes involved in the glutamine synthetase-glutamine oxoglutarate aminotransferase (GS-GOGAT) pathway. The increased E1 degradation rate with acetic acid or humic acid in the background can also be attributed to the up-regulation of IlvE. Results from this study can help predict and explain E1 biodegradation kinetics under various environmental conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Lange, Vinzenz; Malmström, Johan A; Didion, John; King, Nichole L; Johansson, Björn P; Schäfer, Juliane; Rameseder, Jonathan; Wong, Chee-Hong; Deutsch, Eric W; Brusniak, Mi-Youn; Bühlmann, Peter; Björck, Lars; Domon, Bruno; Aebersold, Ruedi
2008-08-01
In many studies, particularly in the field of systems biology, it is essential that identical protein sets are precisely quantified in multiple samples such as those representing differentially perturbed cell states. The high degree of reproducibility required for such experiments has not been achieved by classical mass spectrometry-based proteomics methods. In this study we describe the implementation of a targeted quantitative approach by which predetermined protein sets are first identified and subsequently quantified at high sensitivity reliably in multiple samples. This approach consists of three steps. First, the proteome is extensively mapped out by multidimensional fractionation and tandem mass spectrometry, and the data generated are assembled in the PeptideAtlas database. Second, based on this proteome map, peptides uniquely identifying the proteins of interest, proteotypic peptides, are selected, and multiple reaction monitoring (MRM) transitions are established and validated by MS2 spectrum acquisition. This process of peptide selection, transition selection, and validation is supported by a suite of software tools, TIQAM (Targeted Identification for Quantitative Analysis by MRM), described in this study. Third, the selected target protein set is quantified in multiple samples by MRM. Applying this approach we were able to reliably quantify low abundance virulence factors from cultures of the human pathogen Streptococcus pyogenes exposed to increasing amounts of plasma. The resulting quantitative protein patterns enabled us to clearly define the subset of virulence proteins that is regulated upon plasma exposure.
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He, Dan; Xie, Xiao; Yang, Fan; Zhang, Heng; Su, Haomiao; Ge, Yun; Song, Haiping; Chen, Peng R
2017-11-13
A genetically encoded, multifunctional photocrosslinker was developed for quantitative and comparative proteomics. By bearing a bioorthogonal handle and a releasable linker in addition to its photoaffinity warhead, this probe enables the enrichment of transient and low-abundance prey proteins after intracellular photocrosslinking and prey-bait separation, which can be subject to stable isotope dimethyl labeling and mass spectrometry analysis. This quantitative strategy (termed isoCAPP) allowed a comparative proteomic approach to be adopted to identify the proteolytic substrates of an E. coli protease-chaperone dual machinery DegP. Two newly identified substrates were subsequently confirmed by proteolysis experiments. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Affordable proteomics: the two-hybrid systems.
Gillespie, Marc
2003-06-01
Numerous proteomic methodologies exist, but most require a heavy investment in expertise and technology. This puts these approaches out of reach for many laboratories and small companies, rarely allowing proteomics to be used as a pilot approach for biomarker or target identification. Two proteomic approaches, 2D gel electrophoresis and the two-hybrid systems, are currently available to most researchers. The two-hybrid systems, though accommodating to large-scale experiments, were originally designed as practical screens, that by comparison to current proteomics tools were small-scale, affordable and technically feasible. The screens rapidly generated data, identifying protein interactions that were previously uncharacterized. The foundation for a two-hybrid proteomic investigation can be purchased as separate kits from a number of companies. The true power of the technique lies not in its affordability, but rather in its portability. The two-hybrid system puts proteomics back into laboratories where the output of the screens can be evaluated by researchers with experience in the particular fields of basic research, cancer biology, toxicology or drug development.
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Dynamic quantitative proteomics characterization of TNF-α-induced necroptosis.
Wang, Yang; Huang, Zhi-Hao; Li, Yang-Jia; He, Gui-Wei; Yu, Ru-Yuan; Yang, Jie; Liu, Wan-Ting; Li, Bin; He, Qing-Yu
2016-12-01
Emerging evidence suggested that necroptosis has essential functions in many human inflammatory diseases, but the molecular mechanisms of necroptosis remain unclear. Here, we employed SILAC quantitatively dynamic proteomics to compare the protein changes during TNF-α-induced necroptosis at different time points in murine fibrosarcoma L929 cells with caspase-8 deficiency, and then performed the systematical analysis on the signaling networks involved in the progress using bioinformatics methods. Our results showed that a total of 329, 421 and 378 differentially expressed proteins were detected at three stages of necroptosis, respectively. Gene ontology and ingenuity pathway analysis (IPA) revealed that the proteins regulated at early stages of necroptosis (2, 6 h) were mainly involved in mitochondria dysfunction, oxidative phosphorylation and Nrf-2 signaling, while the expression levels of the proteins related to ubiquitin, Nrf-2, and NF-κB pathways were found to have changes at last stages of necroptosis (6, 18 h). Taken together, we demonstrated for the first time that dysfunction of mitochondria and ubiquitin-proteasome signaling contributed to the initiation and execution of necroptosis. These findings may provide clues for the identification of important regulators in necroptosis and the development of novel therapeutic strategies for the related diseases.
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Targeted Proteomics Approach for Precision Plant Breeding.
Chawade, Aakash; Alexandersson, Erik; Bengtsson, Therese; Andreasson, Erik; Levander, Fredrik
2016-02-05
Selected reaction monitoring (SRM) is a targeted mass spectrometry technique that enables precise quantitation of hundreds of peptides in a single run. This technique provides new opportunities for multiplexed protein biomarker measurements. For precision plant breeding, DNA-based markers have been used extensively, but the potential of protein biomarkers has not been exploited. In this work, we developed an SRM marker panel with assays for 104 potato (Solanum tuberosum) peptides selected using univariate and multivariate statistics. Thereafter, using random forest classification, the prediction markers were identified for Phytopthora infestans resistance in leaves, P. infestans resistance in tubers, and plant yield in potato leaf secretome samples. The results suggest that the marker panel has the predictive potential for three traits, two of which have no commercial DNA markers so far. Furthermore, the marker panel was also tested and found to be applicable to potato clones not used during the marker development. The proposed workflow is thus a proof-of-concept for targeted proteomics as an efficient readout in accelerated breeding for complex and agronomically important traits.
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Li, J-Y; Chen, X; Hosseini Moghaddam, S H; Chen, M; Wei, H; Zhong, B-X
2009-10-01
The shotgun approach has gained considerable acknowledgement in recent years as a dominant strategy in proteomics. We observed a dramatic increase of specific protein spots in two-dimensional electrophoresis (2-DE) gels of the silkworm (Bombyx mori) embryo at labrum appearance, a characteristic stage during embryonic development of silkworm which is involved with temperature increase by silkworm raiser. We employed shotgun liquid chromatography tandem mass spectrometry (LC-MS/MS) technology to analyse the proteome of B. mori embryos at this stage. A total of 2168 proteins were identified with an in-house database. Approximately 47% of them had isoelectric point (pI) values distributed theoretically in the range pI 5-7 and approximately 60% of them had molecular weights of 15-45 kDa. Furthermore, 111 proteins had an pI greater than 10 and were difficult to separate by 2-DE. Many important functional proteins related to embryonic development, stress response, DNA transcription/translation, cell growth, proliferation and differentiation, organogenesis and reproduction were identified. Among them proteins related to nervous system development were noticeable. All known heat shock proteins (HSPs) were detected in this developmental stage of B. mori embryo. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed energetic metabolism at this stage. These results were expected to provide more information for proteomic monitoring of the insect embryo and better understanding of the spatiotemporal expression of genes during embryonic developmental processes.
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Bao, Kai; Bostanci, Nagihan; Selevsek, Nathalie; Thurnheer, Thomas; Belibasakis, Georgios N.
2015-01-01
Periodontitis is an infectious disease that causes the inflammatory destruction of the tooth-supporting (periodontal) tissues, caused by polymicrobial biofilm communities growing on the tooth surface. Aggressive periodontitis is strongly associated with the presence of Aggregatibacter actinomycetemcomitans in the subgingival biofilms. Nevertheless, whether and how A. actinomycetemcomitans orchestrates molecular changes within the biofilm is unclear. The aim of this work was to decipher the interactions between A. actinomycetemcomitans and other bacterial species in a multi-species biofilm using proteomic analysis. An in vitro 10-species “subgingival” biofilm model, or its derivative that included additionally A. actinomycetemcomitans, were anaerobically cultivated on hydroxyapatite discs for 64 h. When present, A. actinomycetemcomitans formed dense intra-species clumps within the biofilm mass, and did not affect the numbers of the other species in the biofilm. Liquid chromatography-tandem mass spectrometry was used to identify the proteomic content of the biofilm lysate. A total of 3225 and 3352 proteins were identified in the biofilm, in presence or absence of A. actinomycetemcomitans, respectively. Label-free quantitative proteomics revealed that 483 out of the 728 quantified bacterial proteins (excluding those of A. actinomycetemcomitans) were accordingly regulated. Interestingly, all quantified proteins from Prevotella intermedia were up-regulated, and most quantified proteins from Campylobacter rectus, Streptococcus anginosus, and Porphyromonas gingivalis were down-regulated in presence of A. actinomycetemcomitans. Enrichment of Gene Ontology pathway analysis showed that the regulated groups of proteins were responsible primarily for changes in the metabolic rate, the ferric iron-binding, and the 5S RNA binding capacities, on the universal biofilm level. While the presence of A. actinomycetemcomitans did not affect the numeric composition or absolute
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A Proteomics Approach to Discover Drought Tolerance Proteins in Wheat Pollen Grain at Meiosis Stage.
Fotovat, Reza; Alikhani, Mehdi; Valizadeh, Mostafa; Mirzaei, Mehdi; Salekdeh, Ghasem H
2017-01-01
Plants reproductive phase, when grain yield and consequently farmers' investment is most in jeopardy, is considered as the most sensitive stage to drought stress. In this study, we aimed to explore the proteomic response of wheat anther at meiosis stage in a drought tolerant, Darab, and susceptible, Shiraz, wheat genotypes. Wheat plants were exposed to drought stress at meiosis stage for four days under controlled environmental conditions. Then, anthers from both genotypes were sampled, and their proteomes were examined via quantitative proteomics analysis. Our results demonstrated that short-term stress at meiosis stage reduced plant seed-setting compared to well-watered plants. This reduction was more pronounced in the susceptible genotype, Shiraz, by 51%, compared to the drought tolerant Darab by 14.3%. Proteome analysis revealed that 60 protein spots were drought responsive, out of which 44 were identified using a mass spectrometer. We observed a dramatic up-regulation of several heat shock proteins, as well as induction of Bet v I allergen family proteins, peroxiredoxin-5, and glutathione transferase with similar abundance in both genotypes. However, the abundance of proteins such as several stress response related proteins, including glutaredoxin, proteasome subunit alpha type 5, and ribosomal proteins showed a different response to drought stress in two genotypes. The differential abundance of proteins in two genotypes may suggest mechanisms by which tolerant genotype cope with drought stress. To the best of our knowledge, this is the first proteome analysis of plant reproductive tissue response to drought stress in wheat and could broaden our insight into plant adaptation to drought stress. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
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ProteoSign: an end-user online differential proteomics statistical analysis platform.
Efstathiou, Georgios; Antonakis, Andreas N; Pavlopoulos, Georgios A; Theodosiou, Theodosios; Divanach, Peter; Trudgian, David C; Thomas, Benjamin; Papanikolaou, Nikolas; Aivaliotis, Michalis; Acuto, Oreste; Iliopoulos, Ioannis
2017-07-03
Profiling of proteome dynamics is crucial for understanding cellular behavior in response to intrinsic and extrinsic stimuli and maintenance of homeostasis. Over the last 20 years, mass spectrometry (MS) has emerged as the most powerful tool for large-scale identification and characterization of proteins. Bottom-up proteomics, the most common MS-based proteomics approach, has always been challenging in terms of data management, processing, analysis and visualization, with modern instruments capable of producing several gigabytes of data out of a single experiment. Here, we present ProteoSign, a freely available web application, dedicated in allowing users to perform proteomics differential expression/abundance analysis in a user-friendly and self-explanatory way. Although several non-commercial standalone tools have been developed for post-quantification statistical analysis of proteomics data, most of them are not end-user appealing as they often require very stringent installation of programming environments, third-party software packages and sometimes further scripting or computer programming. To avoid this bottleneck, we have developed a user-friendly software platform accessible via a web interface in order to enable proteomics laboratories and core facilities to statistically analyse quantitative proteomics data sets in a resource-efficient manner. ProteoSign is available at http://bioinformatics.med.uoc.gr/ProteoSign and the source code at https://github.com/yorgodillo/ProteoSign. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Weckwerth, Wolfram; Baginsky, Sacha; Van Wijk, Klass
2009-12-01
In the past 10 years, we have witnessed remarkable advances in the field of plant molecular biology. The rapid development of proteomic technologies and the speed with which these techniques have been applied to the field have altered our perception of how we can analyze proteins in complex systems. At nearly the same time, the availability of the complete genome for the model plant Arabidopsis thaliana was released; this effort provides an unsurpassed resource for the identification of proteins when researchers use MS to analyze plant samples. Recognizing the growth in this area, the Multinational Arabidopsis Steering Committee (MASC) establishedmore » a subcommittee for A. thaliana proteomics in 2006 with the objective of consolidating databases, technique standards, and experimentally validated candidate genes and functions. Since the establishment of the Multinational Arabidopsis Steering Subcommittee for Proteomics (MASCP), many new approaches and resources have become available. Recently, the subcommittee established a webpage to consolidate this information (www.masc-proteomics.org). It includes links to plant proteomic databases, general information about proteomic techniques, meeting information, a summary of proteomic standards, and other relevant resources. Altogether, this website provides a useful resource for the Arabidopsis proteomics community. In the future, the website will host discussions and investigate the cross-linking of databases. The subcommittee members have extensive experience in arabidopsis proteomics and collectively have produced some of the most extensive proteomics data sets for this model plant (Table S1 in the Supporting Information has a list of resources). The largest collection of proteomics data from a single study in A. thaliana was assembled into an accessible database (AtProteome; http://fgcz-atproteome.unizh.ch/index.php) and was recently published by the Baginsky lab.1 The database provides links to major Arabidopsis
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Falter, Christian; Ellinger, Dorothea; von Hülsen, Behrend; Heim, René; Voigt, Christian A
2015-01-01
The outwardly directed cell wall and associated plasma membrane of epidermal cells represent the first layers of plant defense against intruding pathogens. Cell wall modifications and the formation of defense structures at sites of attempted pathogen penetration are decisive for plant defense. A precise isolation of these stress-induced structures would allow a specific analysis of regulatory mechanism and cell wall adaption. However, methods for large-scale epidermal tissue preparation from the model plant Arabidopsis thaliana, which would allow proteome and cell wall analysis of complete, laser-microdissected epidermal defense structures, have not been provided. We developed the adhesive tape - liquid cover glass technique (ACT) for simple leaf epidermis preparation from A. thaliana, which is also applicable on grass leaves. This method is compatible with subsequent staining techniques to visualize stress-related cell wall structures, which were precisely isolated from the epidermal tissue layer by laser microdissection (LM) coupled to laser pressure catapulting. We successfully demonstrated that these specific epidermal tissue samples could be used for quantitative downstream proteome and cell wall analysis. The development of the ACT for simple leaf epidermis preparation and the compatibility to LM and downstream quantitative analysis opens new possibilities in the precise examination of stress- and pathogen-related cell wall structures in epidermal cells. Because the developed tissue processing is also applicable on A. thaliana, well-established, model pathosystems that include the interaction with powdery mildews can be studied to determine principal regulatory mechanisms in plant-microbe interaction with their potential outreach into crop breeding.
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Falter, Christian; Ellinger, Dorothea; von Hülsen, Behrend; Heim, René; Voigt, Christian A.
2015-01-01
The outwardly directed cell wall and associated plasma membrane of epidermal cells represent the first layers of plant defense against intruding pathogens. Cell wall modifications and the formation of defense structures at sites of attempted pathogen penetration are decisive for plant defense. A precise isolation of these stress-induced structures would allow a specific analysis of regulatory mechanism and cell wall adaption. However, methods for large-scale epidermal tissue preparation from the model plant Arabidopsis thaliana, which would allow proteome and cell wall analysis of complete, laser-microdissected epidermal defense structures, have not been provided. We developed the adhesive tape – liquid cover glass technique (ACT) for simple leaf epidermis preparation from A. thaliana, which is also applicable on grass leaves. This method is compatible with subsequent staining techniques to visualize stress-related cell wall structures, which were precisely isolated from the epidermal tissue layer by laser microdissection (LM) coupled to laser pressure catapulting. We successfully demonstrated that these specific epidermal tissue samples could be used for quantitative downstream proteome and cell wall analysis. The development of the ACT for simple leaf epidermis preparation and the compatibility to LM and downstream quantitative analysis opens new possibilities in the precise examination of stress- and pathogen-related cell wall structures in epidermal cells. Because the developed tissue processing is also applicable on A. thaliana, well-established, model pathosystems that include the interaction with powdery mildews can be studied to determine principal regulatory mechanisms in plant–microbe interaction with their potential outreach into crop breeding. PMID:25870605
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Proteomic Approaches and Identification of Novel Therapeutic Targets for Alcoholism
Gorini, Giorgio; Adron Harris, R; Dayne Mayfield, R
2014-01-01
Recent studies have shown that gene regulation is far more complex than previously believed and does not completely explain changes at the protein level. Therefore, the direct study of the proteome, considerably different in both complexity and dynamicity to the genome/transcriptome, has provided unique insights to an increasing number of researchers. During the past decade, extraordinary advances in proteomic techniques have changed the way we can analyze the composition, regulation, and function of protein complexes and pathways underlying altered neurobiological conditions. When combined with complementary approaches, these advances provide the contextual information for decoding large data sets into meaningful biologically adaptive processes. Neuroproteomics offers potential breakthroughs in the field of alcohol research by leading to a deeper understanding of how alcohol globally affects protein structure, function, interactions, and networks. The wealth of information gained from these advances can help pinpoint relevant biomarkers for early diagnosis and improved prognosis of alcoholism and identify future pharmacological targets for the treatment of this addiction. PMID:23900301
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Optimized approaches for quantification of drug transporters in tissues and cells by MRM proteomics.
Prasad, Bhagwat; Unadkat, Jashvant D
2014-07-01
Drug transporter expression in tissues (in vivo) usually differs from that in cell lines used to measure transporter activity (in vitro). Therefore, quantification of transporter expression in tissues and cell lines is important to develop scaling factor for in vitro to in vivo extrapolation (IVIVE) of transporter-mediated drug disposition. Since traditional immunoquantification methods are semiquantitative, targeted proteomics is now emerging as a superior method to quantify proteins, including membrane transporters. This superiority is derived from the selectivity, precision, accuracy, and speed of analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Moreover, LC-MS/MS proteomics has broader applicability because it does not require selective antibodies for individual proteins. There are a number of recent research and review papers that discuss the use of LC-MS/MS for transporter quantification. Here, we have compiled from the literature various elements of MRM proteomics to provide a comprehensive systematic strategy to quantify drug transporters. This review emphasizes practical aspects and challenges in surrogate peptide selection, peptide qualification, peptide synthesis and characterization, membrane protein isolation, protein digestion, sample preparation, LC-MS/MS parameter optimization, method validation, and sample analysis. In particular, bioinformatic tools used in method development and sample analysis are discussed in detail. Various pre-analytical and analytical sources of variability that should be considered during transporter quantification are highlighted. All these steps are illustrated using P-glycoprotein (P-gp) as a case example. Greater use of quantitative transporter proteomics will lead to a better understanding of the role of drug transporters in drug disposition.
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Ordureau, Alban; Sarraf, Shireen A; Duda, David M; Heo, Jin-Mi; Jedrychowski, Mark P; Sviderskiy, Vladislav O; Olszewski, Jennifer L; Koerber, James T; Xie, Tiao; Beausoleil, Sean A; Wells, James A; Gygi, Steven P; Schulman, Brenda A; Harper, J Wade
2014-11-06
Phosphorylation is often used to promote protein ubiquitylation, yet we rarely understand quantitatively how ligase activation and ubiquitin (UB) chain assembly are integrated with phosphoregulation. Here we employ quantitative proteomics and live-cell imaging to dissect individual steps in the PINK1 kinase-PARKIN UB ligase mitochondrial control pathway disrupted in Parkinson's disease. PINK1 plays a dual role by phosphorylating PARKIN on its UB-like domain and poly-UB chains on mitochondria. PARKIN activation by PINK1 produces canonical and noncanonical UB chains on mitochondria, and PARKIN-dependent chain assembly is required for accumulation of poly-phospho-UB (poly-p-UB) on mitochondria. In vitro, PINK1 directly activates PARKIN's ability to assemble canonical and noncanonical UB chains and promotes association of PARKIN with both p-UB and poly-p-UB. Our data reveal a feedforward mechanism that explains how PINK1 phosphorylation of both PARKIN and poly-UB chains synthesized by PARKIN drives a program of PARKIN recruitment and mitochondrial ubiquitylation in response to mitochondrial damage. Copyright © 2014 Elsevier Inc. All rights reserved.
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Oxidative stress in aging: advances in proteomic approaches.
Ortuño-Sahagún, Daniel; Pallàs, Mercè; Rojas-Mayorquín, Argelia E
2014-01-01
Aging is a gradual, complex process in which cells, tissues, organs, and the whole organism itself deteriorate in a progressive and irreversible manner that, in the majority of cases, implies pathological conditions that affect the individual's Quality of Life (QOL). Although extensive research efforts in recent years have been made, the anticipation of aging and prophylactic or treatment strategies continue to experience major limitations. In this review, the focus is essentially on the compilation of the advances generated by cellular expression profile analysis through proteomics studies (two-dimensional [2D] electrophoresis and mass spectrometry [MS]), which are currently used as an integral approach to study the aging process. Additionally, the relevance of the oxidative stress factors is discussed. Emphasis is placed on postmitotic tissues, such as neuronal, muscular, and red blood cells, which appear to be those most frequently studied with respect to aging. Additionally, models for the study of aging are discussed in a number of organisms, such as Caenorhabditis elegans, senescence-accelerated probe-8 mice (SAMP8), naked mole-rat (Heterocephalus glaber), and the beagle canine. Proteomic studies in specific tissues and organisms have revealed the extensive involvement of reactive oxygen species (ROS) and oxidative stress in aging.
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The Proteome of Native Adult Müller Glial Cells From Murine Retina*
Hauser, Alexandra; Lepper, Marlen Franziska; Mayo, Rebecca
2016-01-01
To date, the proteomic profiling of Müller cells, the dominant macroglia of the retina, has been hampered because of the absence of suitable enrichment methods. We established a novel protocol to isolate native, intact Müller cells from adult murine retinae at excellent purity which retain in situ morphology and are well suited for proteomic analyses. Two different strategies of sample preparation - an in StageTips (iST) and a subcellular fractionation approach including cell surface protein profiling were used for quantitative liquid chromatography-mass spectrometry (LC-MSMS) comparing Müller cell-enriched to depleted neuronal fractions. Pathway enrichment analyses on both data sets enabled us to identify Müller cell-specific functions which included focal adhesion kinase signaling, signal transduction mediated by calcium as second messenger, transmembrane neurotransmitter transport and antioxidant activity. Pathways associated with RNA processing, cellular respiration and phototransduction were enriched in the neuronal subpopulation. Proteomic results were validated for selected Müller cell genes by quantitative real time PCR, confirming the high expression levels of numerous members of the angiogenic and anti-inflammatory annexins and antioxidant enzymes (e.g. paraoxonase 2, peroxiredoxin 1, 4 and 6). Finally, the significant enrichment of antioxidant proteins in Müller cells was confirmed by measurements on vital retinal cells using the oxidative stress indicator CM-H2DCFDA. In contrast to photoreceptors or bipolar cells, Müller cells were most efficiently protected against H2O2-induced reactive oxygen species formation, which is in line with the protein repertoire identified in the proteomic profiling. Our novel approach to isolate intact glial cells from adult retina in combination with proteomic profiling enabled the identification of novel Müller glia specific proteins, which were validated as markers and for their functional impact in glial
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Hansmeier, Nicole; Buttigieg, Josef; Kumar, Pankaj; Pelle, Shaneen; Choi, Kyoo Yoon; Kopriva, David; Chao, Tzu-Chiao
2018-01-05
Atherosclerosis is a chronic inflammatory disease with complex pathobiology and one of the most common causes of cardiovascular events. The process is characterized by complex vascular remodeling processes that require the actions of numerous proteins. The composition of atherosclerotic plaque is increasingly recognized as a major factor governing the occurrence of cardiovascular or neurological symptoms. To gain deeper insights into the composition of atherosclerotic plaques, we created quantitative proteome profiles of advanced plaque tissues of six male patients undergoing carotid endarterectomy for stroke prevention. Using a quantitative, data-independent proteome approach, we identified 4181 proteins with an average protein coverage of 45%. An analysis of the quantitative composition of the tissue revealed key players of vascular remodeling processes. Moreover, compared with proximal arterial tissue, 20 proteins in mature plaques were enriched, whereas 52 proteins were found in lower quantities. Among the proteins with increased abundance were prominent extracellular matrix proteins such as biglycan and lumican, whereas cytoskeletal markers for contractile smooth muscle cells (SMCs) were decreased. Taken together, this study provides the most comprehensive quantitative assessment of mature human plaque tissue to date, which indicates a central role of SMCs in the structure of advanced atherosclerotic plaques.
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Yu, Jia-Lu; Song, Qi-Fang; Xie, Zhi-Wei; Jiang, Wen-Hui; Chen, Jia-Hui; Fan, Hui-Feng; Xie, Ya-Ping; Lu, Gen
2017-09-25
Mycoplasma pneumoniae (MP) is a leading cause of community-acquired pneumonia in children and young adults. Although MP pneumonia is usually benign and self-limited, in some cases it can develop into life-threating refractory MP pneumonia (RMPP). However, the pathogenesis of RMPP is poorly understood. The identification and characterization of proteins related to RMPP could provide a proof of principle to facilitate appropriate diagnostic and therapeutic strategies for treating paients with MP. In this study, we used a quantitative proteomic technique (iTRAQ) to analyze MP-related proteins in serum samples from 5 patients with RMPP, 5 patients with non-refractory MP pneumonia (NRMPP), and 5 healthy children. Functional classification, sub-cellular localization, and protein interaction network analysis were carried out based on protein annotation through evolutionary relationship (PANTHER) and Cytoscape analysis. A total of 260 differentially expressed proteins were identified in the RMPP and NRMPP groups. Compared to the control group, the NRMPP and RMPP groups showed 134 (70 up-regulated and 64 down-regulated) and 126 (63 up-regulated and 63 down-regulated) differentially expressed proteins, respectively. The complex functional classification and protein interaction network of the identified proteins reflected the complex pathogenesis of RMPP. Our study provides the first comprehensive proteome map of RMPP-related proteins from MP pneumonia. These profiles may be useful as part of a diagnostic panel, and the identified proteins provide new insights into the pathological mechanisms underlying RMPP.
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Biochemical Markers of Brain Injury: An Integrated Proteomics-Based Approach
2005-02-01
Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author( s ) and should not be construed as an official...Brain Injury: An Integrated DAMD17-03-1-0066 Proteomics-Based Approach 6. AUTHOR( S ) Ronald L. Hayes, Ph.D. 7. PERFORMING ORGANIZATION NAME( S ) AND ADDRESS...10. SPONSORING / MONITORING AGENCY NAME( S ) AND ADDRESS(ES) AGENCY REPORT NUMBER U.S. Army Medical Research and Materiel Command Fort Detrick
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Hong, Haifa; Ye, Lincai; Chen, Huiwen; Xia, Yu; Liu, Yue; Liu, Jinfen; Lu, Yanan; Zhang, Haibo
2015-08-01
We aimed to evaluate global changes in protein expression associated with patency by undertaking proteomic analysis of human constricted and patent ductus arteriosus (DA). Ten constricted and 10 patent human DAs were excised from infants with ductal-dependent heart disease during surgery. Using isobaric tags for relative and absolute quantitation-based quantitative proteomics, 132 differentially expressed proteins were identified. Of 132 proteins, voltage-gated sodium channel 1.3 (SCN3A), myosin 1d (Myo1d), Rho GTPase activating protein 26 (ARHGAP26), and retinitis pigmentosa 1 (RP1) were selected for validation by Western blot and quantitative real-time polymerase chain reaction analyses. Significant upregulation of SCN3A, Myo1d, and RP1 messenger RNA, and protein levels was observed in the patent DA group (all P ≤ 0.048). ARHGAP26 messenger RNA and protein levels were decreased in patent DA tissue (both P ≤ 0.018). Immunohistochemistry analysis revealed that Myo1d, ARHGAP26, and RP1 were specifically expressed in the subendothelial region of constricted DAs; however, diffuse expression of these proteins was noted in the patent group. Proteomic analysis revealed global changes in the expression of proteins that regulate oxygen sensing, ion channels, smooth muscle cell migration, nervous system, immune system, and metabolism, suggesting a basis for the systemic regulation of DA patency by diverse signaling pathways, which will be confirmed in further studies.
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Abdallah, Cosette; Valot, Benoit; Guillier, Christelle; Mounier, Arnaud; Balliau, Thierry; Zivy, Michel; van Tuinen, Diederik; Renaut, Jenny; Wipf, Daniel; Dumas-Gaudot, Eliane; Recorbet, Ghislaine
2014-08-28
Arbuscular mycorrhizal (AM) symbiosis that associates roots of most land plants with soil-borne fungi (Glomeromycota), is characterized by reciprocal nutritional benefits. Fungal colonization of plant roots induces massive changes in cortical cells where the fungus differentiates an arbuscule, which drives proliferation of the plasma membrane. Despite the recognized importance of membrane proteins in sustaining AM symbiosis, the root microsomal proteome elicited upon mycorrhiza still remains to be explored. In this study, we first examined the qualitative composition of the root membrane proteome of Medicago truncatula after microsome enrichment and subsequent in depth analysis by GeLC-MS/MS. The results obtained highlighted the identification of 1226 root membrane protein candidates whose cellular and functional classifications predispose plastids and protein synthesis as prevalent organelle and function, respectively. Changes at the protein abundance level between the membrane proteomes of mycorrhizal and nonmycorrhizal roots were further monitored by spectral counting, which retrieved a total of 96 proteins that displayed a differential accumulation upon AM symbiosis. Besides the canonical markers of the periarbuscular membrane, new candidates supporting the importance of membrane trafficking events during mycorrhiza establishment/functioning were identified, including flotillin-like proteins. The data have been deposited to the ProteomeXchange with identifier PXD000875. During arbuscular mycorrhizal symbiosis, one of the most widespread mutualistic associations in nature, the endomembrane system of plant roots is believed to undergo qualitative and quantitative changes in order to sustain both the accommodation process of the AM fungus within cortical cells and the exchange of nutrients between symbionts. Large-scale GeLC-MS/MS proteomic analysis of the membrane fractions from mycorrhizal and nonmycorrhizal roots of M. truncatula coupled to spectral counting
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Di Girolamo, Francesco; Righetti, Pier Giorgio; Soste, Martin; Feng, Yuehan; Picotti, Paola
2013-08-26
Systems biology studies require the capability to quantify with high precision proteins spanning a broad range of abundances across multiple samples. However, the broad range of protein expression in cells often precludes the detection of low-abundance proteins. Different sample processing techniques can be applied to increase proteome coverage. Among these, combinatorial (hexa)peptide ligand libraries (CPLLs) bound to solid matrices have been used to specifically capture and detect low-abundance proteins in complex samples. To assess whether CPLL capture can be applied in systems biology studies involving the precise quantitation of proteins across a multitude of samples, we evaluated its performance across the whole range of protein abundances in Saccharomyces cerevisiae. We used selected reaction monitoring assays for a set of target proteins covering a broad abundance range to quantitatively evaluate the precision of the approach and its capability to detect low-abundance proteins. Replicated CPLL-isolates showed an average variability of ~10% in the amount of the isolated proteins. The high reproducibility of the technique was not dependent on the abundance of the protein or the amount of beads used for the capture. However, the protein-to-bead ratio affected the enrichment of specific proteins. We did not observe a normalization effect of CPLL beads on protein abundances. However, CPLLs enriched for and depleted specific sets of proteins and thus changed the abundances of proteins from a whole proteome extract. This allowed the identification of ~400 proteins otherwise undetected in an untreated sample, under the experimental conditions used. CPLL capture is thus a useful tool to increase protein identifications in proteomic experiments, but it should be coupled to the analysis of untreated samples, to maximize proteome coverage. Our data also confirms that CPLL capture is reproducible and can be confidently used in quantitative proteomic experiments
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Owens, Rebecca A.; Hammel, Stephen; Sheridan, Kevin J.; Jones, Gary W.; Doyle, Sean
2014-01-01
A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of secondary metabolites in Aspergillus fumigatus. Here, implementation of a shotgun proteomic strategy led to the identification of non-redundant mycelial proteins (n = 414) from A. fumigatus including proteins typically under-represented in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Indirect identification of secondary metabolite cluster expression was also achieved, with proteins (n = 18) from LaeA-regulated clusters detected, including GliT encoded within the gliotoxin biosynthetic cluster. Biochemical analysis then revealed that gliotoxin significantly attenuates H2O2-induced oxidative stress in A. fumigatus (p>0.0001), confirming observations from proteomics data. A complementary 2-D/LC-MS/MS approach further elucidated significantly increased abundance (p<0.05) of proliferating cell nuclear antigen (PCNA), NADH-quinone oxidoreductase and the gliotoxin oxidoreductase GliT, along with significantly attenuated abundance (p<0.05) of a heat shock protein, an oxidative stress protein and an autolysis-associated chitinase, when gliotoxin and H2O2 were present, compared to H2O2 alone. Moreover, gliotoxin exposure significantly reduced the abundance of selected proteins (p<0.05) involved in de novo purine biosynthesis. Significantly elevated abundance (p<0.05) of a key enzyme, xanthine-guanine phosphoribosyl transferase Xpt1, utilised in purine salvage, was observed in the presence of H2O2 and gliotoxin. This work provides new insights into the A. fumigatus proteome and experimental strategies, plus mechanistic data pertaining to gliotoxin functionality in the organism. PMID:25198175
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Candidate-based proteomics in the search for biomarkers of cardiovascular disease
Anderson, Leigh
2005-01-01
The key concept of proteomics (looking at many proteins at once) opens new avenues in the search for clinically useful biomarkers of disease, treatment response and ageing. As the number of proteins that can be detected in plasma or serum (the primary clinical diagnostic samples) increases towards 1000, a paradoxical decline has occurred in the number of new protein markers approved for diagnostic use in clinical laboratories. This review explores the limitations of current proteomics protein discovery platforms, and proposes an alternative approach, applicable to a range of biological/physiological problems, in which quantitative mass spectrometric methods developed for analytical chemistry are employed to measure limited sets of candidate markers in large sets of clinical samples. A set of 177 candidate biomarker proteins with reported associations to cardiovascular disease and stroke are presented as a starting point for such a ‘directed proteomics’ approach. PMID:15611012
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Proteomic analysis of Medulloblastoma reveals functional biology with translational potential.
Rivero-Hinojosa, Samuel; Lau, Ling San; Stampar, Mojca; Staal, Jerome; Zhang, Huizhen; Gordish-Dressman, Heather; Northcott, Paul A; Pfister, Stefan M; Taylor, Michael D; Brown, Kristy J; Rood, Brian R
2018-06-07
Genomic characterization has begun to redefine diagnostic classifications of cancers. However, it remains a challenge to infer disease phenotypes from genomic alterations alone. To help realize the promise of genomics, we have performed a quantitative proteomics investigation using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and 41 tissue samples spanning the 4 genomically based subgroups of medulloblastoma and control cerebellum. We have identified and quantitated thousands of proteins across these groups and find that we are able to recapitulate the genomic subgroups based upon subgroup restricted and differentially abundant proteins while also identifying subgroup specific protein isoforms. Integrating our proteomic measurements with genomic data, we calculate a poor correlation between mRNA and protein abundance. Using EPIC 850 k methylation array data on the same tissues, we also investigate the influence of copy number alterations and DNA methylation on the proteome in an attempt to characterize the impact of these genetic features on the proteome. Reciprocally, we are able to use the proteome to identify which genomic alterations result in altered protein abundance and thus are most likely to impact biology. Finally, we are able to assemble protein-based pathways yielding potential avenues for clinical intervention. From these, we validate the EIF4F cap-dependent translation pathway as a novel druggable pathway in medulloblastoma. Thus, quantitative proteomics complements genomic platforms to yield a more complete understanding of functional tumor biology and identify novel therapeutic targets for medulloblastoma.
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Proteomic analysis of enterotoxigenic Escherichia coli (ETEC) in neutral and alkaline conditions.
Gonzales-Siles, Lucia; Karlsson, Roger; Kenny, Diarmuid; Karlsson, Anders; Sjöling, Åsa
2017-01-07
Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and travelers to endemic areas. Secretion of the heat labile AB 5 toxin (LT) is induced by alkaline conditions. In this study, we determined the surface proteome of ETEC exposed to alkaline conditions (pH 9) as compared to neutral conditions (pH 7) using a LPI Hexalane FlowCell combined with quantitative proteomics. Relative quantitation with isobaric labeling (TMT) was used to compare peptide abundance and their corresponding proteins in multiple samples at MS/MS level. For protein identification and quantification samples were analyzed using either a 1D-LCMS or a 2D-LCMS approach. Strong up-regulation of the ATP synthase operon encoding F1Fo ATP synthase and down-regulation of proton pumping proteins NuoF, NuoG, Ndh and WrbA were detected among proteins involved in regulating the proton and electron transport under alkaline conditions. Reduced expression of proteins involved in osmotic stress was found at alkaline conditions while the Sec-dependent transport over the inner membrane and outer membrane protein proteins such as OmpA and the β-Barrel Assembly Machinery (BAM) complex were up-regulated. ETEC exposed to alkaline environments express a specific proteome profile characterized by up-regulation of membrane proteins and secretion of LT toxin. Alkaline microenvironments have been reported close to the intestinal epithelium and the alkaline proteome may hence represent a better view of ETEC during infection.
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NASA Astrophysics Data System (ADS)
Zhao, Xiaozheng; Huffman, Kenneth E.; Fujimoto, Junya; Canales, Jamie Rodriguez; Girard, Luc; Nie, Guangjun; Heymach, John V.; Wistuba, Igacio I.; Minna, John D.; Yu, Yonghao
2017-10-01
With recent advances in understanding the genomic underpinnings and oncogenic drivers of pathogenesis in different subtypes, it is increasingly clear that proper pretreatment diagnostics are essential for the choice of appropriate treatment options for non-small cell lung cancer (NSCLC). Tumor tissue preservation in optimal cutting temperature (OCT) compound is commonly used in the surgical suite. However, proteins recovered from OCT-embedded specimens pose a challenge for LC-MS/MS experiments, due to the large amounts of polymers present in OCT. Here we present a simple workflow for whole proteome analysis of OCT-embedded NSCLC tissue samples, which involves a simple trichloroacetic acid precipitation step. Comparisons of protein recovery between frozen versus OCT-embedded tissue showed excellent consistency with more than 9200 proteins identified. Using an isobaric labeling strategy, we quantified more than 5400 proteins in tumor versus normal OCT-embedded core needle biopsy samples. Gene ontology analysis indicated that a number of proliferative as well as squamous cell carcinoma (SqCC) marker proteins were overexpressed in the tumor, consistent with the patient's pathology based diagnosis of "poorly differentiated SqCC". Among the most downregulated proteins in the tumor sample, we noted a number of proteins with potential immunomodulatory functions. Finally, interrogation of the aberrantly expressed proteins using a candidate approach and cross-referencing with publicly available databases led to the identification of potential druggable targets in DNA replication and DNA damage repair pathways. We conclude that our approach allows LC-MS/MS proteomic analyses on OCT-embedded lung cancer specimens, opening the way to bring powerful proteomics into the clinic. [Figure not available: see fulltext.
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Recent insights into plant-virus interactions through proteomic analysis.
Di Carli, Mariasole; Benvenuto, Eugenio; Donini, Marcello
2012-10-05
Plant viruses represent a major threat for a wide range of host species causing severe losses in agricultural practices. The full comprehension of mechanisms underlying events of virus-host plant interaction is crucial to devise novel plant resistance strategies. Until now, functional genomics studies in plant-virus interaction have been limited mainly on transcriptomic analysis. Only recently are proteomic approaches starting to provide important contributions to this area of research. Classical two-dimensional electrophoresis (2-DE) coupled to mass spectrometry (MS) is still the most widely used platform in plant proteome analysis, although in the last years the application of quantitative "second generation" proteomic techniques (such as differential in gel electrophoresis, DIGE, and gel-free protein separation methods) are emerging as more powerful analytical approaches. Apparently simple, plant-virus interactions reveal a really complex pathophysiological context, in which resistance, defense and susceptibility, and direct virus-induced reactions interplay to trigger expression responses of hundreds of genes. Given that, this review is specifically focused on comparative proteome-based studies on pathogenesis of several viral genera, including some of the most important and widespread plant viruses of the genus Tobamovirus, Sobemovirus, Cucumovirus and Potyvirus. In all, this overview reveals a widespread repression of proteins associated with the photosynthetic apparatus, while energy metabolism/protein synthesis and turnover are typically up-regulated, indicating a major redirection of cell metabolism. Other common features include the modulation of metabolisms concerning sugars, cell wall, and reactive oxigen species as well as pathogenesis-related (PR) proteins. The fine-tuning between plant development and antiviral defense mechanisms determines new patterns of regulation of common metabolic pathways. By offering a 360-degree view of protein modulation
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Arntzen, Magnus Ø; Thiede, Bernd
2012-02-01
Apoptosis is the most commonly described form of programmed cell death, and dysfunction is implicated in a large number of human diseases. Many quantitative proteome analyses of apoptosis have been performed to gain insight in proteins involved in the process. This resulted in large and complex data sets that are difficult to evaluate. Therefore, we developed the ApoptoProteomics database for storage, browsing, and analysis of the outcome of large scale proteome analyses of apoptosis derived from human, mouse, and rat. The proteomics data of 52 publications were integrated and unified with protein annotations from UniProt-KB, the caspase substrate database homepage (CASBAH), and gene ontology. Currently, more than 2300 records of more than 1500 unique proteins were included, covering a large proportion of the core signaling pathways of apoptosis. Analysis of the data set revealed a high level of agreement between the reported changes in directionality reported in proteomics studies and expected apoptosis-related function and may disclose proteins without a current recognized involvement in apoptosis based on gene ontology. Comparison between induction of apoptosis by the intrinsic and the extrinsic apoptotic signaling pathway revealed slight differences. Furthermore, proteomics has significantly contributed to the field of apoptosis in identifying hundreds of caspase substrates. The database is available at http://apoptoproteomics.uio.no.
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Application of survival analysis methodology to the quantitative analysis of LC-MS proteomics data.
Tekwe, Carmen D; Carroll, Raymond J; Dabney, Alan R
2012-08-01
Protein abundance in quantitative proteomics is often based on observed spectral features derived from liquid chromatography mass spectrometry (LC-MS) or LC-MS/MS experiments. Peak intensities are largely non-normal in distribution. Furthermore, LC-MS-based proteomics data frequently have large proportions of missing peak intensities due to censoring mechanisms on low-abundance spectral features. Recognizing that the observed peak intensities detected with the LC-MS method are all positive, skewed and often left-censored, we propose using survival methodology to carry out differential expression analysis of proteins. Various standard statistical techniques including non-parametric tests such as the Kolmogorov-Smirnov and Wilcoxon-Mann-Whitney rank sum tests, and the parametric survival model and accelerated failure time-model with log-normal, log-logistic and Weibull distributions were used to detect any differentially expressed proteins. The statistical operating characteristics of each method are explored using both real and simulated datasets. Survival methods generally have greater statistical power than standard differential expression methods when the proportion of missing protein level data is 5% or more. In particular, the AFT models we consider consistently achieve greater statistical power than standard testing procedures, with the discrepancy widening with increasing missingness in the proportions. The testing procedures discussed in this article can all be performed using readily available software such as R. The R codes are provided as supplemental materials. ctekwe@stat.tamu.edu.
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Karp, Natasha A; Feret, Renata; Rubtsov, Denis V; Lilley, Kathryn S
2008-03-01
2-DE is an important tool in quantitative proteomics. Here, we compare the deep purple (DP) system with DIGE using both a traditional and the SameSpots approach to gel analysis. Missing values in the traditional approach were found to be a significant issue for both systems. SameSpots attempts to address the missing value problem. SameSpots was found to increase the proportion of low volume data for DP but not for DIGE. For all the analysis methods applied in this study, the assumptions of parametric tests were met. Analysis of the same images gave significantly lower noise with SameSpots (over traditional) for DP, but no difference for DIGE. We propose that SameSpots gave lower noise with DP due to the stabilisation of the spot area by the common spot outline, but this was not seen with DIGE due to the co-detection process which stabilises the area selected. For studies where measurement of small abundance changes is required, a cost-benefit analysis highlights that DIGE was significantly cheaper regardless of the analysis methods. For studies analysing large changes, DP with SameSpots could be an effective alternative to DIGE but this will be dependent on the biological noise of the system under investigation.
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Accurate, Sensitive, and Precise Multiplexed Proteomics Using the Complement Reporter Ion Cluster
Sonnett, Matthew; Yeung, Eyan; Wuhr, Martin
2018-03-09
We present that quantitative analysis of proteomes across multiple time points, organelles, and perturbations is essential for understanding both fundamental biology and disease states. The development of isobaric tags (e.g. TMT) have enabled the simultaneous measurement of peptide abundances across several different conditions. These multiplexed approaches are promising in principle because of advantages in throughput and measurement quality. However, in practice existing multiplexing approaches suffer from key limitations. In its simple implementation (TMT-MS2), measurements are distorted by chemical noise leading to poor measurement accuracy. The current state-of-the-art (TMT-MS3) addresses this, but requires specialized quadrupole-iontrap-Orbitrap instrumentation. The complement reporter ion approachmore » (TMTc) produces high accuracy measurements and is compatible with many more instruments, like quadrupole-Orbitraps. However, the required deconvolution of the TMTc cluster leads to poor measurement precision. Here, we introduce TMTc+, which adds the modeling of the MS2-isolation step into the deconvolution algorithm. The resulting measurements are comparable in precision to TMT-MS3/MS2. The improved duty cycle, and lower filtering requirements make TMTc+ more sensitive than TMT-MS3 and comparable with TMT-MS2. At the same time, unlike TMT-MS2, TMTc+ is exquisitely able to distinguish signal from chemical noise even outperforming TMT-MS3. Lastly, we compare TMTc+ to quantitative label-free proteomics of total HeLa lysate and find that TMTc+ quantifies 7.8k versus 3.9k proteins in a 5-plex sample. At the same time the median coefficient of variation improves from 13% to 4%. Furthermore, TMTc+ advances quantitative proteomics by enabling accurate, sensitive, and precise multiplexed experiments on more commonly used instruments.« less
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Accurate, Sensitive, and Precise Multiplexed Proteomics Using the Complement Reporter Ion Cluster
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sonnett, Matthew; Yeung, Eyan; Wuhr, Martin
We present that quantitative analysis of proteomes across multiple time points, organelles, and perturbations is essential for understanding both fundamental biology and disease states. The development of isobaric tags (e.g. TMT) have enabled the simultaneous measurement of peptide abundances across several different conditions. These multiplexed approaches are promising in principle because of advantages in throughput and measurement quality. However, in practice existing multiplexing approaches suffer from key limitations. In its simple implementation (TMT-MS2), measurements are distorted by chemical noise leading to poor measurement accuracy. The current state-of-the-art (TMT-MS3) addresses this, but requires specialized quadrupole-iontrap-Orbitrap instrumentation. The complement reporter ion approachmore » (TMTc) produces high accuracy measurements and is compatible with many more instruments, like quadrupole-Orbitraps. However, the required deconvolution of the TMTc cluster leads to poor measurement precision. Here, we introduce TMTc+, which adds the modeling of the MS2-isolation step into the deconvolution algorithm. The resulting measurements are comparable in precision to TMT-MS3/MS2. The improved duty cycle, and lower filtering requirements make TMTc+ more sensitive than TMT-MS3 and comparable with TMT-MS2. At the same time, unlike TMT-MS2, TMTc+ is exquisitely able to distinguish signal from chemical noise even outperforming TMT-MS3. Lastly, we compare TMTc+ to quantitative label-free proteomics of total HeLa lysate and find that TMTc+ quantifies 7.8k versus 3.9k proteins in a 5-plex sample. At the same time the median coefficient of variation improves from 13% to 4%. Furthermore, TMTc+ advances quantitative proteomics by enabling accurate, sensitive, and precise multiplexed experiments on more commonly used instruments.« less
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Wheat proteomics: proteome modulation and abiotic stress acclimation
Komatsu, Setsuko; Kamal, Abu H. M.; Hossain, Zahed
2014-01-01
Cellular mechanisms of stress sensing and signaling represent the initial plant responses to adverse conditions. The development of high-throughput “Omics” techniques has initiated a new era of the study of plant molecular strategies for adapting to environmental changes. However, the elucidation of stress adaptation mechanisms in plants requires the accurate isolation and characterization of stress-responsive proteins. Because the functional part of the genome, namely the proteins and their post-translational modifications, are critical for plant stress responses, proteomic studies provide comprehensive information about the fine-tuning of cellular pathways that primarily involved in stress mitigation. This review summarizes the major proteomic findings related to alterations in the wheat proteomic profile in response to abiotic stresses. Moreover, the strengths and weaknesses of different sample preparation techniques, including subcellular protein extraction protocols, are discussed in detail. The continued development of proteomic approaches in combination with rapidly evolving bioinformatics tools and interactive databases will facilitate understanding of the plant mechanisms underlying stress tolerance. PMID:25538718
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Iwamoto, Noriko; Shimada, Takashi
2018-05-01
Since the turn of the century, mass spectrometry (MS) technologies have continued to improve dramatically, and advanced strategies that were impossible a decade ago are increasingly becoming available. The basic characteristics behind these advancements are MS resolution, quantitative accuracy, and information science for appropriate data processing. The spectral data from MS contain various types of information. The benefits of improving the resolution of MS data include accurate molecular structural-derived information, and as a result, we can obtain a refined biomolecular structure determination in a sequential and large-scale manner. Moreover, in MS data, not only accurate structural information but also the generated ion amount plays an important rule. This progress has greatly contributed a research field that captures biological events as a system by comprehensively tracing the various changes in biomolecular dynamics. The sequential changes of proteome expression in biological pathways are very essential, and the amounts of the changes often directly become the targets of drug discovery or indicators of clinical efficacy. To take this proteomic approach, it is necessary to separate the individual MS spectra derived from each biomolecule in the complexed biological samples. MS itself is not so infinite to perform the all peak separation, and we should consider improving the methods for sample processing and purification to make them suitable for injection into MS. The above-described characteristics can only be achieved using MS with any analytical instrument. Moreover, MS is expected to be applied and expand into many fields, not only basic life sciences but also forensic medicine, plant sciences, materials, and natural products. In this review, we focus on the technical fundamentals and future aspects of the strategies for accurate structural identification, structure-indicated quantitation, and on the challenges for pharmacokinetics of high
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Wang, Shichun; Jiang, Tianlun; Fan, Yahan; Zhao, Shuming
2018-03-29
Cryopreservation can slow down the metabolism and decrease the risk of bacterial contamination. But, chilled platelets (PLTs) show a reduced period in circulation due to the rapid clearance by hepatic cells or spleen macrophages after transfusion. The deleterious changes that PLTs undergo are mainly considered the result of PLT protein variation. However, the basis for proteomic variation of stored PLTs remains poorly understood. Besides count, activation markers (CD62P and Annexin V), and aggregation, we used quantitative mass spectrometry to create the first comprehensive and quantitative human PLT proteome of samples stored at different temperatures (22°C, 10°C and -80°C). We found different conditions caused different platelet storage lesion (PSL). PLT count was decreased no matter at what temperature stored. PLTs viability at low temperature dropped by 21.78% and 11.21%, respectively, as compared 10.26% at room temperature, there were no significant differences between the storage methods. Membrane expression of CD62P gradually increased in all groups especially stored at 22°C up to 40% and 10°C up to 30%. However, exposure of PS on the PLT membrane was below 1% in every group. The PLT proteome showed there were 575 and 454 potential proteins identified by general iTRAQ analysis and phosphorylation iTRAQ a nalysis, respectively, among them, 33 common differentially expressed proteins caused by storage time and 44 caused by storage temperature Especially, membrane-bound proteins (such as FERMT3, STX4, MYL9 and TAGLN2) played key roles in PLT storage lesion. The pathways "Endocytosis", "Fc gamma R-mediated phagocytosis" and "Regulation of actin cytoskeleton" were affected predominantly by storage time. And the pathways "SNARE interactions in vesicular transport" and "Vasopressin-regulated water reabsorption" were affected by cold storage in our study. Proteomic results can help us to understand PLT biochemistry and physiology and thus unravel the mechanisms
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Dineshram, Ramadoss; Chandramouli, Kondethimmanahalli; Ko, Ginger Wai Kuen; Zhang, Huoming; Qian, Pei-Yuan; Ravasi, Timothy; Thiyagarajan, Vengatesen
2016-06-01
The metamorphosis of planktonic larvae of the Pacific oyster (Crassostrea gigas) underpins their complex life-history strategy by switching on the molecular machinery required for sessile life and building calcite shells. Metamorphosis becomes a survival bottleneck, which will be pressured by different anthropogenically induced climate change-related variables. Therefore, it is important to understand how metamorphosing larvae interact with emerging climate change stressors. To predict how larvae might be affected in a future ocean, we examined changes in the proteome of metamorphosing larvae under multiple stressors: decreased pH (pH 7.4), increased temperature (30 °C), and reduced salinity (15 psu). Quantitative protein expression profiling using iTRAQ-LC-MS/MS identified more than 1300 proteins. Decreased pH had a negative effect on metamorphosis by down-regulating several proteins involved in energy production, metabolism, and protein synthesis. However, warming switched on these down-regulated pathways at pH 7.4. Under multiple stressors, cell signaling, energy production, growth, and developmental pathways were up-regulated, although metamorphosis was still reduced. Despite the lack of lethal effects, significant physiological responses to both individual and interacting climate change related stressors were observed at proteome level. The metamorphosing larvae of the C. gigas population in the Yellow Sea appear to have adequate phenotypic plasticity at the proteome level to survive in future coastal oceans, but with developmental and physiological costs. © 2016 John Wiley & Sons Ltd.
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Yang, Yongxin; Zhao, Xiaowei; Yu, Shumin; Cao, Suizhong
2015-02-01
Yak (Bos grunniens) is an important natural resource in mountainous regions. To date, few studies have addressed the differences in the protein profiles of yak colostrum and milk. We used quantitative proteomics to compare the protein profiles of whey from yak colostrum and milk. Milk samples were collected from 21 yaks after calving (1 and 28 d). Whey protein profiles were generated through isobaric tag for relative and absolute quantification (iTRAQ)-labelled proteomics. We identified 183 proteins in milk whey; of these, the expression levels of 86 proteins differed significantly between the whey from colostrum and milk. Haemoglobin expression showed the greatest change; its levels were significantly higher in the whey from colostrum than in mature milk whey. Functional analysis revealed that many of the differentially expressed proteins were associated with biological regulation and response to stimuli. Further, eight differentially expressed proteins involved in the complement and coagulation cascade pathway were enriched in milk whey. These findings add to the general understanding of the protein composition of yak milk, suggest potential functions of the differentially expressed proteins, and provide novel information on the role of colostral components in calf survival. © 2014 Society of Chemical Industry.
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Skates, Steven J.; Gillette, Michael A.; LaBaer, Joshua; Carr, Steven A.; Anderson, N. Leigh; Liebler, Daniel C.; Ransohoff, David; Rifai, Nader; Kondratovich, Marina; Težak, Živana; Mansfield, Elizabeth; Oberg, Ann L.; Wright, Ian; Barnes, Grady; Gail, Mitchell; Mesri, Mehdi; Kinsinger, Christopher R.; Rodriguez, Henry; Boja, Emily S.
2014-01-01
Protein biomarkers are needed to deepen our understanding of cancer biology and to improve our ability to diagnose, monitor and treat cancers. Important analytical and clinical hurdles must be overcome to allow the most promising protein biomarker candidates to advance into clinical validation studies. Although contemporary proteomics technologies support the measurement of large numbers of proteins in individual clinical specimens, sample throughput remains comparatively low. This problem is amplified in typical clinical proteomics research studies, which routinely suffer from a lack of proper experimental design, resulting in analysis of too few biospecimens to achieve adequate statistical power at each stage of a biomarker pipeline. To address this critical shortcoming, a joint workshop was held by the National Cancer Institute (NCI), National Heart, Lung and Blood Institute (NHLBI), and American Association for Clinical Chemistry (AACC), with participation from the U.S. Food and Drug Administration (FDA). An important output from the workshop was a statistical framework for the design of biomarker discovery and verification studies. Herein, we describe the use of quantitative clinical judgments to set statistical criteria for clinical relevance, and the development of an approach to calculate biospecimen sample size for proteomic studies in discovery and verification stages prior to clinical validation stage. This represents a first step towards building a consensus on quantitative criteria for statistical design of proteomics biomarker discovery and verification research. PMID:24063748
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Skates, Steven J; Gillette, Michael A; LaBaer, Joshua; Carr, Steven A; Anderson, Leigh; Liebler, Daniel C; Ransohoff, David; Rifai, Nader; Kondratovich, Marina; Težak, Živana; Mansfield, Elizabeth; Oberg, Ann L; Wright, Ian; Barnes, Grady; Gail, Mitchell; Mesri, Mehdi; Kinsinger, Christopher R; Rodriguez, Henry; Boja, Emily S
2013-12-06
Protein biomarkers are needed to deepen our understanding of cancer biology and to improve our ability to diagnose, monitor, and treat cancers. Important analytical and clinical hurdles must be overcome to allow the most promising protein biomarker candidates to advance into clinical validation studies. Although contemporary proteomics technologies support the measurement of large numbers of proteins in individual clinical specimens, sample throughput remains comparatively low. This problem is amplified in typical clinical proteomics research studies, which routinely suffer from a lack of proper experimental design, resulting in analysis of too few biospecimens to achieve adequate statistical power at each stage of a biomarker pipeline. To address this critical shortcoming, a joint workshop was held by the National Cancer Institute (NCI), National Heart, Lung, and Blood Institute (NHLBI), and American Association for Clinical Chemistry (AACC) with participation from the U.S. Food and Drug Administration (FDA). An important output from the workshop was a statistical framework for the design of biomarker discovery and verification studies. Herein, we describe the use of quantitative clinical judgments to set statistical criteria for clinical relevance and the development of an approach to calculate biospecimen sample size for proteomic studies in discovery and verification stages prior to clinical validation stage. This represents a first step toward building a consensus on quantitative criteria for statistical design of proteomics biomarker discovery and verification research.
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Manzanares-Miralles, Lara; Sarikaya-Bayram, Özlem; Smith, Elizabeth B; Dolan, Stephen K; Bayram, Özgür; Jones, Gary W; Doyle, Sean
2016-01-10
Gliotoxin (GT) is a redox-active metabolite, produced by Aspergillus fumigatus, which inhibits the growth of other fungi. Here we demonstrate how Aspergillus niger responds to GT exposure. Quantitative proteomics revealed that GT dysregulated the abundance of 378 proteins including those involved in methionine metabolism and induced de novo abundance of two S-adenosylmethionine (SAM)-dependent methyltransferases. Increased abundance of enzymes S-adenosylhomocysteinase (p=0.0018) required for homocysteine generation from S-adenosylhomocysteine (SAH), and spermidine synthase (p=0.0068), involved in the recycling of Met, was observed. Analysis of Met-related metabolites revealed significant increases in the levels of Met and adenosine, in correlation with proteomic data. Methyltransferase MT-II is responsible for bisthiobis(methylthio)gliotoxin (BmGT) formation, deletion of MT-II abolished BmGT formation and led to increased GT sensitivity in A. niger. Proteomic analysis also revealed that GT exposure also significantly (p<0.05) increased hydrolytic enzyme abundance, including glycoside hydrolases (n=22) and peptidases (n=16). We reveal that in an attempt to protect against the detrimental affects of GT, methyltransferase-mediated GT thiomethylation alters cellular pathways involving Met and SAM, with consequential dysregulation of hydrolytic enzyme abundance in A. niger. Thus, it provides new opportunities to exploit the response of GT-naïve fungi to GT. Copyright © 2015 Elsevier B.V. All rights reserved.
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Proteomic dataset of the sea urchin Paracentrotus lividus adhesive organs and secreted adhesive
Lebesgue, Nicolas; da Costa, Gonçalo; Ribeiro, Raquel Mesquita; Ribeiro-Silva, Cristina; Martins, Gabriel G.; Matranga, Valeria; Scholten, Arjen; Cordeiro, Carlos; Heck, Albert J.R.; Santos, Romana
2016-01-01
Sea urchins have specialized adhesive organs called tube feet, which mediate strong but reversible adhesion. Tube feet are composed by a disc, producing adhesive and de-adhesive secretions for substratum attachment, and a stem for movement. After detachment the secreted adhesive remains bound to the substratum as a footprint. Recently, a label-free quantitative proteomic approach coupled with the latest mass-spectrometry technology was used to analyze the differential proteome of Paracentrotus lividus adhesive organ, comparing protein expression levels in the tube feet adhesive part (the disc) versus the non-adhesive part (the stem), and also to profile the proteome of the secreted adhesive (glue). This data article contains complementary figures and results related to the research article “Deciphering the molecular mechanisms underlying sea urchin reversible adhesion: a quantitative proteomics approach” (Lebesgue et al., 2016) [1]. Here we provide a dataset of 1384 non-redundant proteins, their fragmented peptides and expression levels, resultant from the analysis of the tube feet differential proteome. Of these, 163 highly over-expressed tube feet disc proteins (>3-fold), likely representing the most relevant proteins for sea urchin reversible adhesion, were further annotated in order to determine the potential functions. In addition, we provide a dataset of 611 non-redundant proteins identified in the secreted adhesive proteome, as well as their functional annotation and grouping in 5 major protein groups related with adhesive exocytosis, and microbial protection. This list was further analyzed to identify the most abundant protein groups and pinpoint putative adhesive proteins, such as Nectin, the most abundant adhesive protein in sea urchin glue. The obtained data uncover the key proteins involved in sea urchins reversible adhesion, representing a step forward to the development of new wet-effective bio-inspired adhesives. PMID:27182547
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Gao, Hua-Jun; Chen, Ya-Jing; Zuo, Duo; Xiao, Ming-Ming; Li, Ying; Guo, Hua; Zhang, Ning; Chen, Rui-Bing
2015-01-01
Objective Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Methods Lectin affinity chromatography (LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography (LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. Results A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain (FGG), FOS-like antigen 2 (FOSL2), and α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. Conclusion A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC. PMID:26487969
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Proteomics-based compositional analysis of complex cellulase-hemicellulase mixtures.
Chundawat, Shishir P S; Lipton, Mary S; Purvine, Samuel O; Uppugundla, Nirmal; Gao, Dahai; Balan, Venkatesh; Dale, Bruce E
2011-10-07
Efficient deconstruction of cellulosic biomass to fermentable sugars for fuel and chemical production is accomplished by a complex mixture of cellulases, hemicellulases, and accessory enzymes (e.g., >50 extracellular proteins). Cellulolytic enzyme mixtures, produced industrially mostly using fungi like Trichoderma reesei, are poorly characterized in terms of their protein composition and its correlation to hydrolytic activity on cellulosic biomass. The secretomes of commercial glycosyl hydrolase-producing microbes was explored using a proteomics approach with high-throughput quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here, we show that proteomics-based spectral counting approach is a reasonably accurate and rapid analytical technique that can be used to determine protein composition of complex glycosyl hydrolase mixtures that also correlates with the specific activity of individual enzymes present within the mixture. For example, a strong linear correlation was seen between Avicelase activity and total cellobiohydrolase content. Reliable, quantitative and cheaper analytical methods that provide insight into the cellulosic biomass degrading fungal and bacterial secretomes would lead to further improvements toward commercialization of plant biomass-derived fuels and chemicals.
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Large Scale Proteomic Data and Network-Based Systems Biology Approaches to Explore the Plant World.
Di Silvestre, Dario; Bergamaschi, Andrea; Bellini, Edoardo; Mauri, PierLuigi
2018-06-03
The investigation of plant organisms by means of data-derived systems biology approaches based on network modeling is mainly characterized by genomic data, while the potential of proteomics is largely unexplored. This delay is mainly caused by the paucity of plant genomic/proteomic sequences and annotations which are fundamental to perform mass-spectrometry (MS) data interpretation. However, Next Generation Sequencing (NGS) techniques are contributing to filling this gap and an increasing number of studies are focusing on plant proteome profiling and protein-protein interactions (PPIs) identification. Interesting results were obtained by evaluating the topology of PPI networks in the context of organ-associated biological processes as well as plant-pathogen relationships. These examples foreshadow well the benefits that these approaches may provide to plant research. Thus, in addition to providing an overview of the main-omic technologies recently used on plant organisms, we will focus on studies that rely on concepts of module, hub and shortest path, and how they can contribute to the plant discovery processes. In this scenario, we will also consider gene co-expression networks, and some examples of integration with metabolomic data and genome-wide association studies (GWAS) to select candidate genes will be mentioned.
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Quantitative Peptidomics with Five-plex Reductive Methylation labels
NASA Astrophysics Data System (ADS)
Tashima, Alexandre K.; Fricker, Lloyd D.
2017-12-01
Quantitative peptidomics and proteomics often use chemical tags to covalently modify peptides with reagents that differ in the number of stable isotopes, allowing for quantitation of the relative peptide levels in the original sample based on the peak height of each isotopic form. Different chemical reagents have been used as tags for quantitative peptidomics and proteomics, and all have strengths and weaknesses. One of the simplest approaches uses formaldehyde and sodium cyanoborohydride to methylate amines, converting primary and secondary amines into tertiary amines. Up to five different isotopic forms can be generated, depending on the isotopic forms of formaldehyde and cyanoborohydride reagents, allowing for five-plex quantitation. However, the mass difference between each of these forms is only 1 Da per methyl group incorporated into the peptide, and for many peptides there is substantial overlap from the natural abundance of 13C and other isotopes. In this study, we calculated the contribution from the natural isotopes for 26 native peptides and derived equations to correct the peak intensities. These equations were applied to data from a study using human embryonic kidney HEK293T cells in which five replicates were treated with 100 nM vinblastine for 3 h and compared with five replicates of cells treated with control medium. The correction equations brought the replicates to the expected 1:1 ratios and revealed significant decreases in levels of 21 peptides upon vinblastine treatment. These equations enable accurate quantitation of small changes in peptide levels using the reductive methylation labeling approach. [Figure not available: see fulltext.
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Quantitative Peptidomics with Five-plex Reductive Methylation labels
NASA Astrophysics Data System (ADS)
Tashima, Alexandre K.; Fricker, Lloyd D.
2018-05-01
Quantitative peptidomics and proteomics often use chemical tags to covalently modify peptides with reagents that differ in the number of stable isotopes, allowing for quantitation of the relative peptide levels in the original sample based on the peak height of each isotopic form. Different chemical reagents have been used as tags for quantitative peptidomics and proteomics, and all have strengths and weaknesses. One of the simplest approaches uses formaldehyde and sodium cyanoborohydride to methylate amines, converting primary and secondary amines into tertiary amines. Up to five different isotopic forms can be generated, depending on the isotopic forms of formaldehyde and cyanoborohydride reagents, allowing for five-plex quantitation. However, the mass difference between each of these forms is only 1 Da per methyl group incorporated into the peptide, and for many peptides there is substantial overlap from the natural abundance of 13C and other isotopes. In this study, we calculated the contribution from the natural isotopes for 26 native peptides and derived equations to correct the peak intensities. These equations were applied to data from a study using human embryonic kidney HEK293T cells in which five replicates were treated with 100 nM vinblastine for 3 h and compared with five replicates of cells treated with control medium. The correction equations brought the replicates to the expected 1:1 ratios and revealed significant decreases in levels of 21 peptides upon vinblastine treatment. These equations enable accurate quantitation of small changes in peptide levels using the reductive methylation labeling approach. [Figure not available: see fulltext.
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Fungal proteomics: from identification to function.
Doyle, Sean
2011-08-01
Some fungi cause disease in humans and plants, while others have demonstrable potential for the control of insect pests. In addition, fungi are also a rich reservoir of therapeutic metabolites and industrially useful enzymes. Detailed analysis of fungal biochemistry is now enabled by multiple technologies including protein mass spectrometry, genome and transcriptome sequencing and advances in bioinformatics. Yet, the assignment of function to fungal proteins, encoded either by in silico annotated, or unannotated genes, remains problematic. The purpose of this review is to describe the strategies used by many researchers to reveal protein function in fungi, and more importantly, to consolidate the nomenclature of 'unknown function protein' as opposed to 'hypothetical protein' - once any protein has been identified by protein mass spectrometry. A combination of approaches including comparative proteomics, pathogen-induced protein expression and immunoproteomics are outlined, which, when used in combination with a variety of other techniques (e.g. functional genomics, microarray analysis, immunochemical and infection model systems), appear to yield comprehensive and definitive information on protein function in fungi. The relative advantages of proteomic, as opposed to transcriptomic-only, analyses are also described. In the future, combined high-throughput, quantitative proteomics, allied to transcriptomic sequencing, are set to reveal much about protein function in fungi. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
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Proteomics of filamentous fungi.
Kim, Yonghyun; Nandakumar, M P; Marten, Mark R
2007-09-01
Proteomic analysis, defined here as the global assessment of cellular proteins expressed in a particular biological state, is a powerful tool that can provide a systematic understanding of events at the molecular level. Proteomic studies of filamentous fungi have only recently begun to appear in the literature, despite the prevalence of these organisms in the biotechnology industry, and their importance as both human and plant pathogens. Here, we review recent publications that have used a proteomic approach to develop a better understanding of filamentous fungi, highlighting sample preparation methods and whole-cell cytoplasmic proteomics, as well as subproteomics of cell envelope, mitochondrial and secreted proteins.
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Embryology in the era of proteomics.
Katz-Jaffe, Mandy G; McReynolds, Susanna
2013-03-15
Proteomic technologies have begun providing evidence that viable embryos possess unique protein profiles. Some of these potential protein biomarkers have been identified as extracellular and could be used in the development of a noninvasive quantitative method for embryo assessment. The field of assisted reproductive technologies would benefit from defining the human embryonic proteome and secretome, thereby expanding our current knowledge of embryonic cellular processes. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
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Achievements and perspectives of top-down proteomics.
Armirotti, Andrea; Damonte, Gianluca
2010-10-01
Over the last years, top-down (TD) MS has gained a remarkable space in proteomics, rapidly trespassing the limit between a promising approach and a solid, established technique. Several research groups worldwide have implemented TD analysis in their routine work on proteomics, deriving structural information on proteins with the level of accuracy that is impossible to achieve with classical bottom-up approaches. Complete maps of PTMs and assessment of single aminoacid polymorphisms are only a few of the results that can be obtained with this technique. Despite some existing technical and economical limitations, TD analysis is at present the most powerful instrument for MS-based proteomics and its implementation in routine workflow is a rapidly approaching turning point in proteomics. In this review article, the state-of-the-art of TD approach is described along with its major advantages and drawbacks and the most recent trends in TD analysis are discussed. References for all the covered topics are reported in the text, with the aim to support both newcomers and mass spectrometrists already introduced to TD proteomics.
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Identification of Maturation-Specific Proteins by Single-Cell Proteomics of Human Oocytes
Virant-Klun, Irma; Leicht, Stefan; Hughes, Christopher; Krijgsveld, Jeroen
2016-01-01
Oocytes undergo a range of complex processes via oogenesis, maturation, fertilization, and early embryonic development, eventually giving rise to a fully functioning organism. To understand proteome composition and diversity during maturation of human oocytes, here we have addressed crucial aspects of oocyte collection and proteome analysis, resulting in the first proteome and secretome maps of human oocytes. Starting from 100 oocytes collected via a novel serum-free hanging drop culture system, we identified 2,154 proteins, whose function indicate that oocytes are largely resting cells with a proteome that is tailored for homeostasis, cellular attachment, and interaction with its environment via secretory factors. In addition, we have identified 158 oocyte-enriched proteins (such as ECAT1, PIWIL3, NLRP7)1 not observed in high-coverage proteomics studies of other human cell lines or tissues. Exploiting SP3, a novel technology for proteomic sample preparation using magnetic beads, we scaled down proteome analysis to single cells. Despite the low protein content of only ∼100 ng per cell, we consistently identified ∼450 proteins from individual oocytes. When comparing individual oocytes at the germinal vesicle (GV) and metaphase II (MII) stage, we found that the Tudor and KH domain-containing protein (TDRKH) is preferentially expressed in immature oocytes, while Wee2, PCNA, and DNMT1 were enriched in mature cells, collectively indicating that maintenance of genome integrity is crucial during oocyte maturation. This study demonstrates that an innovative proteomics workflow facilitates analysis of single human oocytes to investigate human oocyte biology and preimplantation development. The approach presented here paves the way for quantitative proteomics in other quantity-limited tissues and cell types. Data associated with this study are available via ProteomeXchange with identifier PXD004142. PMID:27215607
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Identification of Maturation-Specific Proteins by Single-Cell Proteomics of Human Oocytes.
Virant-Klun, Irma; Leicht, Stefan; Hughes, Christopher; Krijgsveld, Jeroen
2016-08-01
Oocytes undergo a range of complex processes via oogenesis, maturation, fertilization, and early embryonic development, eventually giving rise to a fully functioning organism. To understand proteome composition and diversity during maturation of human oocytes, here we have addressed crucial aspects of oocyte collection and proteome analysis, resulting in the first proteome and secretome maps of human oocytes. Starting from 100 oocytes collected via a novel serum-free hanging drop culture system, we identified 2,154 proteins, whose function indicate that oocytes are largely resting cells with a proteome that is tailored for homeostasis, cellular attachment, and interaction with its environment via secretory factors. In addition, we have identified 158 oocyte-enriched proteins (such as ECAT1, PIWIL3, NLRP7)(1) not observed in high-coverage proteomics studies of other human cell lines or tissues. Exploiting SP3, a novel technology for proteomic sample preparation using magnetic beads, we scaled down proteome analysis to single cells. Despite the low protein content of only ∼100 ng per cell, we consistently identified ∼450 proteins from individual oocytes. When comparing individual oocytes at the germinal vesicle (GV) and metaphase II (MII) stage, we found that the Tudor and KH domain-containing protein (TDRKH) is preferentially expressed in immature oocytes, while Wee2, PCNA, and DNMT1 were enriched in mature cells, collectively indicating that maintenance of genome integrity is crucial during oocyte maturation. This study demonstrates that an innovative proteomics workflow facilitates analysis of single human oocytes to investigate human oocyte biology and preimplantation development. The approach presented here paves the way for quantitative proteomics in other quantity-limited tissues and cell types. Data associated with this study are available via ProteomeXchange with identifier PXD004142. © 2016 by The American Society for Biochemistry and Molecular Biology
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Arntzen, Magnus Ø.; Thiede, Bernd
2012-01-01
Apoptosis is the most commonly described form of programmed cell death, and dysfunction is implicated in a large number of human diseases. Many quantitative proteome analyses of apoptosis have been performed to gain insight in proteins involved in the process. This resulted in large and complex data sets that are difficult to evaluate. Therefore, we developed the ApoptoProteomics database for storage, browsing, and analysis of the outcome of large scale proteome analyses of apoptosis derived from human, mouse, and rat. The proteomics data of 52 publications were integrated and unified with protein annotations from UniProt-KB, the caspase substrate database homepage (CASBAH), and gene ontology. Currently, more than 2300 records of more than 1500 unique proteins were included, covering a large proportion of the core signaling pathways of apoptosis. Analysis of the data set revealed a high level of agreement between the reported changes in directionality reported in proteomics studies and expected apoptosis-related function and may disclose proteins without a current recognized involvement in apoptosis based on gene ontology. Comparison between induction of apoptosis by the intrinsic and the extrinsic apoptotic signaling pathway revealed slight differences. Furthermore, proteomics has significantly contributed to the field of apoptosis in identifying hundreds of caspase substrates. The database is available at http://apoptoproteomics.uio.no. PMID:22067098
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Zhang, Guosong; Zhang, Jiajia; Wen, Xin; Zhao, Cheng; Zhang, Hongye; Li, Xinru; Yin, Shaowu
2017-09-01
More and more frequently these days, aquatic ecosystems are being stressed by nutrient enrichment, pollutants, and global warming, leading to a serious depletion in oxygen concentrations. Although a sudden, significant lack of oxygen will result in mortality, fishes can have an acute behavior (e.g., an increase in breathing rate, reduction in swimming frequency) and physiology responses (e.g., increase in oxygen delivery, and reduction in oxygen consumption) to hypoxia, which allows them to maintain normal physical activity. Therefore, in order to shed further light on the molecular mechanisms of hypoxia adaptation in fishes, the authors conduct comparative quantitative proteomics on Pelteobagrus vachelli livers using iTRAQ. The research identifies 511 acute hypoxia-responsive proteins in P. vachelli. Furthermore, comparison of several of the diverse key pathways studied (e.g., peroxisome pathway, PPAR signaling pathway, lipid metabolism, glycolysis/gluco-neogenesis, and amino acid metabolism) help to articulate the different mechanisms involved in the hypoxia response of P. vachelli. Data from proteome analysis shows that P. vachelli can have an acute reaction to hypoxia, including detoxification of metabolic by-products and oxidative stress in light of continued metabolic activity (e.g., peroxisomes), an activation in the capacity of catabolism to get more energy (e.g., lipolysis and amino acid catabolism), a depression in the capacity of biosynthesis to reduce energy consumption (e.g., biosynthesis of amino acids and lipids), and a shift in the aerobic and anaerobic contributions to total metabolism. The observed hypoxia-related changes in the liver proteome of the fish can help to understand or can be related to the hypoxia-related response that takes place in similar conditions in the liver or other proteomes of mammals. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Neural Stem Cells (NSCs) and Proteomics*
Shoemaker, Lorelei D.; Kornblum, Harley I.
2016-01-01
Neural stem cells (NSCs) can self-renew and give rise to the major cell types of the CNS. Studies of NSCs include the investigation of primary, CNS-derived cells as well as animal and human embryonic stem cell (ESC)-derived and induced pluripotent stem cell (iPSC)-derived sources. NSCs provide a means with which to study normal neural development, neurodegeneration, and neurological disease and are clinically relevant sources for cellular repair to the damaged and diseased CNS. Proteomics studies of NSCs have the potential to delineate molecules and pathways critical for NSC biology and the means by which NSCs can participate in neural repair. In this review, we provide a background to NSC biology, including the means to obtain them and the caveats to these processes. We then focus on advances in the proteomic interrogation of NSCs. This includes the analysis of posttranslational modifications (PTMs); approaches to analyzing different proteomic compartments, such the secretome; as well as approaches to analyzing temporal differences in the proteome to elucidate mechanisms of differentiation. We also discuss some of the methods that will undoubtedly be useful in the investigation of NSCs but which have not yet been applied to the field. While many proteomics studies of NSCs have largely catalogued the proteome or posttranslational modifications of specific cellular states, without delving into specific functions, some have led to understandings of functional processes or identified markers that could not have been identified via other means. Many challenges remain in the field, including the precise identification and standardization of NSCs used for proteomic analyses, as well as how to translate fundamental proteomics studies to functional biology. The next level of investigation will require interdisciplinary approaches, combining the skills of those interested in the biochemistry of proteomics with those interested in modulating NSC function. PMID:26494823
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Neural Stem Cells (NSCs) and Proteomics.
Shoemaker, Lorelei D; Kornblum, Harley I
2016-02-01
Neural stem cells (NSCs) can self-renew and give rise to the major cell types of the CNS. Studies of NSCs include the investigation of primary, CNS-derived cells as well as animal and human embryonic stem cell (ESC)-derived and induced pluripotent stem cell (iPSC)-derived sources. NSCs provide a means with which to study normal neural development, neurodegeneration, and neurological disease and are clinically relevant sources for cellular repair to the damaged and diseased CNS. Proteomics studies of NSCs have the potential to delineate molecules and pathways critical for NSC biology and the means by which NSCs can participate in neural repair. In this review, we provide a background to NSC biology, including the means to obtain them and the caveats to these processes. We then focus on advances in the proteomic interrogation of NSCs. This includes the analysis of posttranslational modifications (PTMs); approaches to analyzing different proteomic compartments, such the secretome; as well as approaches to analyzing temporal differences in the proteome to elucidate mechanisms of differentiation. We also discuss some of the methods that will undoubtedly be useful in the investigation of NSCs but which have not yet been applied to the field. While many proteomics studies of NSCs have largely catalogued the proteome or posttranslational modifications of specific cellular states, without delving into specific functions, some have led to understandings of functional processes or identified markers that could not have been identified via other means. Many challenges remain in the field, including the precise identification and standardization of NSCs used for proteomic analyses, as well as how to translate fundamental proteomics studies to functional biology. The next level of investigation will require interdisciplinary approaches, combining the skills of those interested in the biochemistry of proteomics with those interested in modulating NSC function. © 2016 by The
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Proteomic Analyses of the Effects of Drugs of Abuse on Monocyte-Derived Mature Dendritic Cells
Reynolds, Jessica L.; Mahajan, Supriya D.; Aalinkeel, Ravikunar; Nair, B.; Sykes, Donald E.; Schwartz, Stanley A.
2010-01-01
Drug abuse has become a global health concern. Understanding how drug abuse modulates the immune system and how the immune system responds to pathogens associated with drug abuse, such hepatitis C virus (HCV) and human immunodeficiency virus (HIV-1), can be assessed by an integrated approach comparing proteomic analyses and quantitation of gene expression. Two-dimensional (2D) difference gel electrophoresis was used to determine the molecular mechanisms underlying the proteomic changes that alter normal biological processes when monocyte-derived mature dendritic cells were treated with cocaine or methamphetamine. Both drugs differentially regulated the expression of several functional classes of proteins including those that modulate apoptosis, protein folding, protein kinase activity, and metabolism and proteins that function as intracellular signal transduction molecules. Proteomic data were validated using a combination of quantitative, real-time PCR and Western blot analyses. These studies will help to identify the molecular mechanisms, including the expression of several functionally important classes of proteins that have emerged as potential mediators of pathogenesis. These proteins may predispose immunocompetent cells, including dendritic cells, to infection with viruses such as HCV and HIV-1, which are associated with drug abuse. PMID:19811410
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Zhu, Ying; Piehowski, Paul D.; Zhao, Rui
Nanoscale or single cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (Nanodroplet Processing in One pot for Trace Samples) platform as a major advance in overall sensitivity. NanoPOTS dramatically enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive LC-MS, nanoPOTS allows identification of ~1500 to ~3,000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runsmore » algorithm of MaxQuant, >3000 proteins were consistently identified from as few as 10 cells. Furthermore, we demonstrate robust quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.« less
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The hemolymph proteome of fed and starved Drosophila larvae.
Handke, Björn; Poernbacher, Ingrid; Goetze, Sandra; Ahrens, Christian H; Omasits, Ulrich; Marty, Florian; Simigdala, Nikiana; Meyer, Imke; Wollscheid, Bernd; Brunner, Erich; Hafen, Ernst; Lehner, Christian F
2013-01-01
The co-operation of specialized organ systems in complex multicellular organisms depends on effective chemical communication. Thus, body fluids (like blood, lymph or intraspinal fluid) contain myriads of signaling mediators apart from metabolites. Moreover, these fluids are also of crucial importance for immune and wound responses. Compositional analyses of human body fluids are therefore of paramount diagnostic importance. Further improving their comprehensiveness should increase our understanding of inter-organ communication. In arthropods, which have trachea for gas exchange and an open circulatory system, the single dominating interstitial fluid is the hemolymph. Accordingly, a detailed analysis of hemolymph composition should provide an especially comprehensive picture of chemical communication and defense in animals. Therefore we used an extensive protein fractionation workflow in combination with a discovery-driven proteomic approach to map out the detectable protein composition of hemolymph isolated from Drosophila larvae. Combined mass spectrometric analysis revealed more than 700 proteins extending far beyond the previously known Drosophila hemolymph proteome. Moreover, by comparing hemolymph isolated from either fed or starved larvae, we provide initial provisional insights concerning compositional changes in response to nutritional state. Storage proteins in particular were observed to be strongly reduced by starvation. Our hemolymph proteome catalog provides a rich basis for data mining, as exemplified by our identification of potential novel cytokines, as well as for future quantitative analyses by targeted proteomics.
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The Hemolymph Proteome of Fed and Starved Drosophila Larvae
Goetze, Sandra; Ahrens, Christian H.; Omasits, Ulrich; Marty, Florian; Simigdala, Nikiana; Meyer, Imke; Wollscheid, Bernd; Brunner, Erich; Hafen, Ernst; Lehner, Christian F.
2013-01-01
The co-operation of specialized organ systems in complex multicellular organisms depends on effective chemical communication. Thus, body fluids (like blood, lymph or intraspinal fluid) contain myriads of signaling mediators apart from metabolites. Moreover, these fluids are also of crucial importance for immune and wound responses. Compositional analyses of human body fluids are therefore of paramount diagnostic importance. Further improving their comprehensiveness should increase our understanding of inter-organ communication. In arthropods, which have trachea for gas exchange and an open circulatory system, the single dominating interstitial fluid is the hemolymph. Accordingly, a detailed analysis of hemolymph composition should provide an especially comprehensive picture of chemical communication and defense in animals. Therefore we used an extensive protein fractionation workflow in combination with a discovery-driven proteomic approach to map out the detectable protein composition of hemolymph isolated from Drosophila larvae. Combined mass spectrometric analysis revealed more than 700 proteins extending far beyond the previously known Drosophila hemolymph proteome. Moreover, by comparing hemolymph isolated from either fed or starved larvae, we provide initial provisional insights concerning compositional changes in response to nutritional state. Storage proteins in particular were observed to be strongly reduced by starvation. Our hemolymph proteome catalog provides a rich basis for data mining, as exemplified by our identification of potential novel cytokines, as well as for future quantitative analyses by targeted proteomics. PMID:23840627
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2011-01-01
Background Nucleolus is the most prominent mammalian organelle within the nucleus which is also the site for ribosomal biogenesis. There have been many reports indicating the involvement of nucleolus in the process of aging. Several proteins related to aging have been shown to localize in the nucleolus, which suggests the role of this organelle in senescence. Results In this study, we used quantitative mass spectrometry to map the flux of proteins into and out of the nucleolus during the induction of senescence in cultured mammalian cells. Changes in the abundance of 344 nucleolar proteins in sodium butyrate-induced senescence in NIH3T3 cells were studied by SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry. Biochemically, we have validated the proteomic results and confirmed that B23 (nucleophosmin) protein was down-regulated, while poly (ADP-ribose) polymerase (PARP) and nuclear DNA helicase II (NDH II/DHX9/RHA) were up-regulated in the nucleolus upon treatment with sodium butyrate. Accumulation of chromatin in the nucleolus was also observed, by both proteomics and microscopy, in sodium butyrate-treated cells. Similar observations were found in other models of senescence, namely, in mitoxantrone- (MTX) treated cells and primary fibroblasts from the Lamin A knockout mice. Conclusion Our data indicate an extensive nuclear organization during senescence and suggest that the redistribution of B23 protein and chromatin can be used as an important marker for senescence. PMID:21835027
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Thekkiniath, Jose; Zabet-Moghaddam, Masoud; Kottapalli, Kameswara Rao; Pasham, Mithun R.; San Francisco, Susan; San Francisco, Michael
2015-01-01
Batrachochytrium dendrobatidis (Bd), a chytrid fungus, has increasingly been implicated as a major factor in the worldwide decline of amphibian populations. The fungus causes chytridiomycosis in susceptible species leading to massive die-offs of adult amphibians. Although Bd infects the keratinized mouthparts of tadpoles and negatively affects foraging behavior, these infections are non-lethal. An important morphogen controlling amphibian metamorphosis is thyroid hormone (T3). Tadpoles may be infected with Bd and the fungus may be exposed to T3 during metamorphosis. We hypothesize that exposure of Bd to T3 may induce the expression of factors associated with host colonization and pathogenicity. We utilized a proteomics approach to better understand the dynamics of the Bd-T3 interaction. Using liquid chromatography-mass spectrometry (LC-MS), we generated a data set of a large number of cytoplasmic and membrane proteins following exposure of Bd to T3. From these data, we identified a total of 263 proteins whose expression was significantly changed following T3 exposure. We provide evidence for expression of an array of proteins that may play key roles in both genomic and non-genomic actions of T3 in Bd. Additionally, our proteomics study shows an increase in several proteins including proteases and a class of uncommon crinkler and crinkler-like effector proteins suggesting their importance in Bd pathogenicity as well as those involved in metabolism and energy transfer, protein fate, transport and stress responses. This approach provides insights into the mechanistic basis of the Bd-amphibian interaction following T3 exposure. PMID:26046527
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Thekkiniath, Jose; Zabet-Moghaddam, Masoud; Kottapalli, Kameswara Rao; Pasham, Mithun R; San Francisco, Susan; San Francisco, Michael
2015-01-01
Batrachochytrium dendrobatidis (Bd), a chytrid fungus, has increasingly been implicated as a major factor in the worldwide decline of amphibian populations. The fungus causes chytridiomycosis in susceptible species leading to massive die-offs of adult amphibians. Although Bd infects the keratinized mouthparts of tadpoles and negatively affects foraging behavior, these infections are non-lethal. An important morphogen controlling amphibian metamorphosis is thyroid hormone (T3). Tadpoles may be infected with Bd and the fungus may be exposed to T3 during metamorphosis. We hypothesize that exposure of Bd to T3 may induce the expression of factors associated with host colonization and pathogenicity. We utilized a proteomics approach to better understand the dynamics of the Bd-T3 interaction. Using liquid chromatography-mass spectrometry (LC-MS), we generated a data set of a large number of cytoplasmic and membrane proteins following exposure of Bd to T3. From these data, we identified a total of 263 proteins whose expression was significantly changed following T3 exposure. We provide evidence for expression of an array of proteins that may play key roles in both genomic and non-genomic actions of T3 in Bd. Additionally, our proteomics study shows an increase in several proteins including proteases and a class of uncommon crinkler and crinkler-like effector proteins suggesting their importance in Bd pathogenicity as well as those involved in metabolism and energy transfer, protein fate, transport and stress responses. This approach provides insights into the mechanistic basis of the Bd-amphibian interaction following T3 exposure.
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Barkla, Bronwyn J.; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar
2009-01-01
To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na+ sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H+-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H+-pump activity. PMID:20028841
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Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells
Lam, Yun W.; Evans, Vanessa C.; Heesom, Kate J.; Lamond, Angus I.; Matthews, David A.
2010-01-01
Adenoviruses replicate primarily in the host cell nucleus, and it is well established that adenovirus infection affects the structure and function of host cell nucleoli in addition to coding for a number of nucleolar targeted viral proteins. Here we used unbiased proteomics methods, including high throughput mass spectrometry coupled with stable isotope labeling by amino acids in cell culture (SILAC) and traditional two-dimensional gel electrophoresis, to identify quantitative changes in the protein composition of the nucleolus during adenovirus infection. Two-dimensional gel analysis revealed changes in six proteins. By contrast, SILAC-based approaches identified 351 proteins with 24 proteins showing at least a 2-fold change after infection. Of those, four were previously reported to have aberrant localization and/or functional relevance during adenovirus infection. In total, 15 proteins identified as changing in amount by proteomics methods were examined in infected cells using confocal microscopy. Eleven of these proteins showed altered patterns of localization in adenovirus-infected cells. Comparing our data with the effects of actinomycin D on the nucleolar proteome revealed that adenovirus infection apparently specifically targets a relatively small subset of nucleolar antigens at the time point examined. PMID:19812395
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Proteomics analysis of the nucleolus in adenovirus-infected cells.
Lam, Yun W; Evans, Vanessa C; Heesom, Kate J; Lamond, Angus I; Matthews, David A
2010-01-01
Adenoviruses replicate primarily in the host cell nucleus, and it is well established that adenovirus infection affects the structure and function of host cell nucleoli in addition to coding for a number of nucleolar targeted viral proteins. Here we used unbiased proteomics methods, including high throughput mass spectrometry coupled with stable isotope labeling by amino acids in cell culture (SILAC) and traditional two-dimensional gel electrophoresis, to identify quantitative changes in the protein composition of the nucleolus during adenovirus infection. Two-dimensional gel analysis revealed changes in six proteins. By contrast, SILAC-based approaches identified 351 proteins with 24 proteins showing at least a 2-fold change after infection. Of those, four were previously reported to have aberrant localization and/or functional relevance during adenovirus infection. In total, 15 proteins identified as changing in amount by proteomics methods were examined in infected cells using confocal microscopy. Eleven of these proteins showed altered patterns of localization in adenovirus-infected cells. Comparing our data with the effects of actinomycin D on the nucleolar proteome revealed that adenovirus infection apparently specifically targets a relatively small subset of nucleolar antigens at the time point examined.
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Oestrus synchronisation and superovulation alter the cervicovaginal mucus proteome of the ewe.
Maddison, Jessie W; Rickard, Jessica P; Bernecic, Naomi C; Tsikis, Guillaume; Soleilhavoup, Clement; Labas, Valerie; Combes-Soia, Lucie; Harichaux, Gregoire; Druart, Xavier; Leahy, Tamara; de Graaf, Simon P
2017-02-23
Although essential for artificial insemination (AI) and MOET (multiple ovulation and embryo transfer), oestrus synchronisation and superovulation are associated with increased female reproductive tract mucus production and altered sperm transport. The effects of such breeding practices on the ovine cervicovaginal (CV) mucus proteome have not been detailed. The aim of this study was to qualitatively and quantitatively investigate the Merino CV mucus proteome in naturally cycling (NAT) ewes at oestrus and mid-luteal phase, and quantitatively compare CV oestrus mucus proteomes of NAT, progesterone synchronised (P4) and superovulated (SOV) ewes. Quantitative analysis revealed 60 proteins were more abundant during oestrus and 127 were more abundant during the luteal phase, with 27 oestrus specific and 40 luteal specific proteins identified. The oestrus proteins most disparate in abundance compared to mid-luteal phase were ceruloplasmin (CP), chitinase-3-like protein 1 (CHI3L1), clusterin (CLU), alkaline phosphatase (ALPL) and mucin-16 (MUC16). Exogenous hormones greatly altered the proteome with 51 and 32 proteins more abundant and 98 and 53 proteins less abundant, in P4 and SOV mucus, respectively when compared to NAT mucus. Investigation of the impact of these proteomic changes on sperm motility and longevity within mucus may help improve sperm transport and fertility following cervical AI. This manuscript is the first to detail the proteome of ovine cervicovaginal mucus using qualitative and quantitative proteomic methods over the oestrous cycle in naturally cycling ewes, and also after application of common oestrus synchronisation and superovulation practices. The investigation of the mucus proteome throughout both the follicular and luteal periods of the oestrous cycle, and also after oestrous synchronisation and superovulation provides information about the endocrine control and the effects that exogenous hormones have on protein expression in the female
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Trends in mass spectrometry instrumentation for proteomics
DOE Office of Scientific and Technical Information (OSTI.GOV)
Smith, Richard D.
2002-12-01
Mass spectrometry has become a primary tool for proteomics due to its capabilities for rapid and sensitive protein identification and quantitation. It is now possible to identify thousands of proteins from microgram sample quantities in a single day and to quantify relative protein abundances. However, the needs for increased capabilities for proteome measurements are immense and are now driving both new strategies and instrument advances. These developments include those based on integration with multi-dimensional liquid separations and high accuracy mass measurements, and promise more than order of magnitude improvements in sensitivity, dynamic range, and throughput for proteomic analyses in themore » near future.« less
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Time-Resolved Proteomic Visualization of Dendrimer Cellular Entry and Trafficking.
Wang, Linna; Yang, Li; Pan, Li; Kadasala, Naveen Reddy; Xue, Liang; Schuster, Robert J; Parker, Laurie L; Wei, Alexander; Tao, W Andy
2015-10-14
Our understanding of the complex cell entry pathways would greatly benefit from a comprehensive characterization of key proteins involved in this dynamic process. Here we devise a novel proteomic strategy named TITAN (Tracing Internalization and TrAfficking of Nanomaterials) to reveal real-time protein-dendrimer interactions using a systems biology approach. Dendrimers functionalized with photoreactive cross-linkers were internalized by HeLa cells and irradiated at set time intervals, then isolated and subjected to quantitative proteomics. In total, 809 interacting proteins cross-linked with dendrimers were determined by TITAN in a detailed temporal manner during dendrimer internalization, traceable to at least two major endocytic mechanisms, clathrin-mediated and caveolar/raft-mediated endocytosis. The direct involvement of the two pathways was further established by the inhibitory effect of dynasore on dendrimer uptake and changes in temporal profiles of key proteins.
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Tian, Xiaoxu; Chen, Lei; Wang, Jiangxin; Qiao, Jianjun; Zhang, Weiwen
2013-01-14
Butanol is a promising biofuel, and recent metabolic engineering efforts have demonstrated the use of photosynthetic cyanobacterial hosts for its production. However, cyanobacteria have very low tolerance to butanol, limiting the economic viability of butanol production from these renewable producing systems. The existing knowledge of molecular mechanism involved in butanol tolerance in cyanobacteria is very limited. To build a foundation necessary to engineer robust butanol-producing cyanobacterial hosts, in this study, the responses of Synechocystis PCC 6803 to butanol were investigated using a quantitative proteomics approach with iTRAQ - LC-MS/MS technologies. The resulting high-quality dataset consisted of 25,347 peptides corresponding to 1452 unique proteins, a coverage of approximately 40% of the predicted proteins in Synechocystis. Comparative quantification of protein abundances led to the identification of 303 differentially regulated proteins by butanol. Annotation and GO term enrichment analysis showed that multiple biological processes were regulated, suggesting that Synechocystis probably employed multiple and synergistic resistance mechanisms in dealing with butanol stress. Notably, the analysis revealed the induction of heat-shock protein and transporters, along with modification of cell membrane and envelope were the major protection mechanisms against butanol. A conceptual cellular model of Synechocystis PCC 6803 responses to butanol stress was constructed to illustrate the putative molecular mechanisms employed to defend against butanol stress. Copyright © 2012 Elsevier B.V. All rights reserved.
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Progress on the HUPO Draft Human Proteome: 2017 Metrics of the Human Proteome Project.
Omenn, Gilbert S; Lane, Lydie; Lundberg, Emma K; Overall, Christopher M; Deutsch, Eric W
2017-12-01
The Human Proteome Organization (HUPO) Human Proteome Project (HPP) continues to make progress on its two overall goals: (1) completing the protein parts list, with an annual update of the HUPO draft human proteome, and (2) making proteomics an integrated complement to genomics and transcriptomics throughout biomedical and life sciences research. neXtProt version 2017-01-23 has 17 008 confident protein identifications (Protein Existence [PE] level 1) that are compliant with the HPP Guidelines v2.1 ( https://hupo.org/Guidelines ), up from 13 664 in 2012-12 and 16 518 in 2016-04. Remaining to be found by mass spectrometry and other methods are 2579 "missing proteins" (PE2+3+4), down from 2949 in 2016. PeptideAtlas 2017-01 has 15 173 canonical proteins, accounting for nearly all of the 15 290 PE1 proteins based on MS data. These resources have extensive data on PTMs, single amino acid variants, and splice isoforms. The Human Protein Atlas v16 has 10 492 highly curated protein entries with tissue and subcellular spatial localization of proteins and transcript expression. Organ-specific popular protein lists have been generated for broad use in quantitative targeted proteomics using SRM-MS or DIA-SWATH-MS studies of biology and disease.
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Salt stress induces changes in the proteomic profile of micropropagated sugarcane shoots
Reis, Ricardo S.; Heringer, Angelo S.; Rangel, Patricia L.; Santa-Catarina, Claudete; Grativol, Clícia; Veiga, Carlos F. M.; Souza-Filho, Gonçalo A.
2017-01-01
Salt stress is one of the most common stresses in agricultural regions worldwide. In particular, sugarcane is affected by salt stress conditions, and no sugarcane cultivar presently show high productivity accompanied by a tolerance to salt stress. Proteomic analysis allows elucidation of the important pathways involved in responses to various abiotic stresses at the biochemical and molecular levels. Thus, this study aimed to analyse the proteomic effects of salt stress in micropropagated shoots of two sugarcane cultivars (CB38-22 and RB855536) using a label-free proteomic approach. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD006075. The RB855536 cultivar is more tolerant to salt stress than CB38-22. A quantitative label-free shotgun proteomic analysis identified 1172 non-redundant proteins, and 1160 of these were observed in both cultivars in the presence or absence of NaCl. Compared with CB38-22, the RB855536 cultivar showed a greater abundance of proteins involved in non-enzymatic antioxidant mechanisms, ion transport, and photosynthesis. Some proteins, such as calcium-dependent protein kinase, photosystem I, phospholipase D, and glyceraldehyde-3-phosphate dehydrogenase, were more abundant in the RB855536 cultivar under salt stress. Our results provide new insights into the response of sugarcane to salt stress, and the changes in the abundance of these proteins might be important for the acquisition of ionic and osmotic homeostasis during exposure to salt stress. PMID:28419154
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Systems Proteomics for Translational Network Medicine
Arrell, D. Kent; Terzic, Andre
2012-01-01
Universal principles underlying network science, and their ever-increasing applications in biomedicine, underscore the unprecedented capacity of systems biology based strategies to synthesize and resolve massive high throughput generated datasets. Enabling previously unattainable comprehension of biological complexity, systems approaches have accelerated progress in elucidating disease prediction, progression, and outcome. Applied to the spectrum of states spanning health and disease, network proteomics establishes a collation, integration, and prioritization algorithm to guide mapping and decoding of proteome landscapes from large-scale raw data. Providing unparalleled deconvolution of protein lists into global interactomes, integrative systems proteomics enables objective, multi-modal interpretation at molecular, pathway, and network scales, merging individual molecular components, their plurality of interactions, and functional contributions for systems comprehension. As such, network systems approaches are increasingly exploited for objective interpretation of cardiovascular proteomics studies. Here, we highlight network systems proteomic analysis pipelines for integration and biological interpretation through protein cartography, ontological categorization, pathway and functional enrichment and complex network analysis. PMID:22896016
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Multiple testing corrections in quantitative proteomics: A useful but blunt tool.
Pascovici, Dana; Handler, David C L; Wu, Jemma X; Haynes, Paul A
2016-09-01
Multiple testing corrections are a useful tool for restricting the FDR, but can be blunt in the context of low power, as we demonstrate by a series of simple simulations. Unfortunately, in proteomics experiments low power can be common, driven by proteomics-specific issues like small effects due to ratio compression, and few replicates due to reagent high cost, instrument time availability and other issues; in such situations, most multiple testing corrections methods, if used with conventional thresholds, will fail to detect any true positives even when many exist. In this low power, medium scale situation, other methods such as effect size considerations or peptide-level calculations may be a more effective option, even if they do not offer the same theoretical guarantee of a low FDR. Thus, we aim to highlight in this article that proteomics presents some specific challenges to the standard multiple testing corrections methods, which should be employed as a useful tool but not be regarded as a required rubber stamp. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Rashid, Muhammad Ibrahim; Naz, Anam; Ali, Amjad; Andleeb, Saadia
2017-07-01
Pseudomonas aeruginosa is among top critical nosocomial infectious agents due to its persistent infections and tendency for acquiring drug resistance mechanisms. To date, there is no vaccine available for this pathogen. We attempted to exploit the genomic and proteomic information of P. aeruginosa though reverse-vaccinology approaches to unveil the prospective vaccine candidates. P. aeruginosa strain PAO1 genome was subjected to sequential prioritization approach following genomic, proteomics and structural analyses. Among, the predicted vaccine candidates: surface components of antibiotic efflux pumps (Q9HY88, PA2837), chaperone-usher pathway components (CupC2, CupB3), penicillin binding protein of bacterial cell wall (PBP1a/mrcA), extracellular component of Type 3 secretory system (PscC) and three uncharacterized secretory proteins (PA0629, PA2822, PA0978) were identified as potential candidates qualifying all the set criteria. These proteins were then analyzed for potential immunogenic surface exposed epitopes. These predicted epitopes may provide a basis for development of a reliable subunit vaccine against P. aeruginosa. Copyright © 2017 Elsevier Inc. All rights reserved.
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Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles
Antrobus, Robin; Hirst, Jennifer; Bhumbra, Gary S.; Kozik, Patrycja; Jackson, Lauren P.; Sahlender, Daniela A.
2012-01-01
Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a “profiling” cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions. PMID:22472443
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How may targeted proteomics complement genomic data in breast cancer?
Guerin, Mathilde; Gonçalves, Anthony; Toiron, Yves; Baudelet, Emilie; Audebert, Stéphane; Boyer, Jean-Baptiste; Borg, Jean-Paul; Camoin, Luc
2017-01-01
Breast cancer (BC) is the most common female cancer in the world and was recently deconstructed in different molecular entities. Although most of the recent assays to characterize tumors at the molecular level are genomic-based, proteins are the actual executors of cellular functions and represent the vast majority of targets for anticancer drugs. Accumulated data has demonstrated an important level of quantitative and qualitative discrepancies between genomic/transcriptomic alterations and their protein counterparts, mostly related to the large number of post-translational modifications. Areas covered: This review will present novel proteomics technologies such as Reverse Phase Protein Array (RPPA) or mass-spectrometry (MS) based approaches that have emerged and that could progressively replace old-fashioned methods (e.g. immunohistochemistry, ELISA, etc.) to validate proteins as diagnostic, prognostic or predictive biomarkers, and eventually monitor them in the routine practice. Expert commentary: These different targeted proteomic approaches, able to complement genomic data in BC and characterize tumors more precisely, will permit to go through a more personalized treatment for each patient and tumor.
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Proteomics for understanding miRNA biology
Huang, Tai-Chung; Pinto, Sneha M.; Pandey, Akhilesh
2013-01-01
MicroRNAs (miRNAs) are small noncoding RNAs that play important roles in posttranscriptional regulation of gene expression. Mature miRNAs associate with the RNA interference silencing complex to repress mRNA translation and/or degrade mRNA transcripts. Mass spectrometry-based proteomics has enabled identification of several core components of the canonical miRNA processing pathway and their posttranslational modifications which are pivotal in miRNA regulatory mechanisms. The use of quantitative proteomic strategies has also emerged as a key technique for experimental identification of miRNA targets by allowing direct determination of proteins whose levels are altered because of translational suppression. This review focuses on the role of proteomics and labeling strategies to understand miRNA biology. PMID:23125164
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Application of proteomics to ecology and population biology.
Karr, T L
2008-02-01
Proteomics is a relatively new scientific discipline that merges protein biochemistry, genome biology and bioinformatics to determine the spatial and temporal expression of proteins in cells, tissues and whole organisms. There has been very little application of proteomics to the fields of behavioral genetics, evolution, ecology and population dynamics, and has only recently been effectively applied to the closely allied fields of molecular evolution and genetics. However, there exists considerable potential for proteomics to impact in areas related to functional ecology; this review will introduce the general concepts and methodologies that define the field of proteomics and compare and contrast the advantages and disadvantages with other methods. Examples of how proteomics can aid, complement and indeed extend the study of functional ecology will be discussed including the main tool of ecological studies, population genetics with an emphasis on metapopulation structure analysis. Because proteomic analyses provide a direct measure of gene expression, it obviates some of the limitations associated with other genomic approaches, such as microarray and EST analyses. Likewise, in conjunction with associated bioinformatics and molecular evolutionary tools, proteomics can provide the foundation of a systems-level integration approach that can enhance ecological studies. It can be envisioned that proteomics will provide important new information on issues specific to metapopulation biology and adaptive processes in nature. A specific example of the application of proteomics to sperm ageing is provided to illustrate the potential utility of the approach.
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2013-01-01
Background Down syndrome (DS), caused by an extra copy of chromosome 21, affects 1 in 750 live births and is characterized by cognitive impairment and a constellation of congenital defects. Currently, little is known about the molecular pathogenesis and no direct genotype-phenotype relationship has yet been confirmed. Since DS amniocytes are expected to have a distinct biological behaviour compared to normal amniocytes, we hypothesize that relative quantification of proteins produced from trisomy and euploid (chromosomally normal) amniocytes will reveal dysregulated molecular pathways. Results Chromosomally normal- and Trisomy 21-amniocytes were quantitatively analyzed by using Stable Isotope Labeling of Amino acids in Cell culture and tandem mass spectrometry. A total of 4919 unique proteins were identified from the supernatant and cell lysate proteome. More specifically, 4548 unique proteins were identified from the lysate, and 91% of these proteins were quantified based on MS/MS spectra ratios of peptides containing isotope-labeled amino acids. A total of 904 proteins showed significant differential expression and were involved in 25 molecular pathways, each containing a minimum of 16 proteins. Sixty of these proteins consistently showed aberrant expression from trisomy 21 affected amniocytes, indicating their potential role in DS pathogenesis. Nine proteins were analyzed with a multiplex selected reaction monitoring assay in an independent set of Trisomy 21-amniocyte samples and two of them (SOD1 and NES) showed a consistent differential expression. Conclusions The most extensive proteome of amniocytes and amniotic fluid has been generated and differentially expressed proteins from amniocytes with Trisomy 21 revealed molecular pathways that seem to be most significantly affected by the presence of an extra copy of chromosome 21. PMID:23394617
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Clark, David J; Fondrie, William E; Yang, Austin; Mao, Li
2016-02-05
Exosomes are 30-100 nm sized membrane vesicles released by cells into the extracellular space that mediate intercellular communication via transfer of proteins and other biological molecules. To better understand the role of these microvesicles in lung carcinogenesis, we employed a Triple SILAC quantitative proteomic strategy to examine the differential protein abundance between exosomes derived from an immortalized normal bronchial epithelial cell line and two non-small cell lung cancer (NSCLC) cell lines harboring distinct activating mutations in the cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) or epidermal growth factor receptor (EGFR). In total, we were able to quantify 721 exosomal proteins derived from the three cell lines. Proteins associated with signal transduction, including EGFR, GRB2 and SRC, were enriched in NSCLC exosomes, and could actively regulate cell proliferation in recipient cells. This study's investigation of the NSCLC exosomal proteome has identified enriched protein cargo that can contribute to lung cancer progression, which may have potential clinical implications in biomarker development for patients with NSCLC. The high mortality associated with lung cancer is a result of late-stage diagnosis of the disease. Current screening techniques used for early detection of lung cancer lack the specificity for accurate diagnosis. Exosomes are nano-sized extracellular vesicles, and the increased abundance of select protein cargo in exosomes derived from cancer cells may be used for diagnostic purposes. In this paper, we applied quantitative proteomic analysis to elucidate abundance differences in exosomal protein cargo between two NSCLC cell lines with distinctive oncogene mutations and an immortalized normal bronchial epithelial cell line. This study revealed proteins associated with cell adhesion, the extracellular matrix, and a variety of signaling molecules were enriched in NSCLC exosomes. The present data reveals
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Li, Qian-Feng; Xiong, Min; Xu, Peng; Huang, Li-Chun; Zhang, Chang-Quan; Liu, Qiao-Quan
2016-01-01
Brassinosteroids (BRs), essential plant-specific steroidal hormones, function in a wide spectrum of plant growth and development events, including seed germination. Rice is not only a monocotyledonous model plant but also one of the most important staple food crops of human beings. Rice seed germination is a decisive event for the next-generation of plant growth and successful seed germination is critical for rice yield. However, little is known about the molecular mechanisms on how BR modulates seed germination in rice. In the present study, we used isobaric tags for relative and absolute quantification (iTRAQ) based proteomic approach to study BR-regulated proteome during the early stage of seed germination. The results showed that more than 800 BR-responsive proteins were identified, including 88 reliable target proteins responsive to stimuli of both BR-deficiency and BR-insensitivity. Moreover, 90% of the 88 target proteins shared a similar expression change pattern. Gene ontology and string analysis indicated that ribosomal structural proteins, as well as proteins involved in protein biosynthesis and carbohydrate metabolisms were highly clustered. These findings not only enrich BR-regulated protein database in rice seeds, but also allow us to gain novel insights into the molecular mechanism of BR regulated seed germination. PMID:27703189
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Recent advances in proteomics of cereals.
Bansal, Monika; Sharma, Madhu; Kanwar, Priyanka; Goyal, Aakash
Cereals contribute a major part of human nutrition and are considered as an integral source of energy for human diets. With genomic databases already available in cereals such as rice, wheat, barley, and maize, the focus has now moved to proteome analysis. Proteomics studies involve the development of appropriate databases based on developing suitable separation and purification protocols, identification of protein functions, and can confirm their functional networks based on already available data from other sources. Tremendous progress has been made in the past decade in generating huge data-sets for covering interactions among proteins, protein composition of various organs and organelles, quantitative and qualitative analysis of proteins, and to characterize their modulation during plant development, biotic, and abiotic stresses. Proteomics platforms have been used to identify and improve our understanding of various metabolic pathways. This article gives a brief review of efforts made by different research groups on comparative descriptive and functional analysis of proteomics applications achieved in the cereal science so far.
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Zhang, Jiang; Guy, Moltu J.; Norman, Holly S.; Chen, Yi-Chen; Xu, Qingge; Dong, Xintong; Guner, Huseyin; Wang, Sijian; Kohmoto, Takushi; Young, Ken H.; Moss, Richard L.; Ge, Ying
2011-01-01
The rapid increase in the prevalence of chronic heart failure (CHF) worldwide underscores an urgent need to identify biomarkers for the early detection of CHF. Post-translational modifications (PTMs) are associated with many critical signaling events during disease progression and thus offer a plethora of candidate biomarkers. We have employed top-down quantitative proteomics methodology for comprehensive assessment of PTMs in whole proteins extracted from normal and diseased tissues. We have systematically analyzed thirty-six clinical human heart tissue samples and identified phosphorylation of cardiac troponin I (cTnI) as a candidate biomarker for CHF. The relative percentages of the total phosphorylated cTnI forms over the entire cTnI populations (%Ptotal) were 56.4±3.5%, 36.9±1.6%, 6.1±2.4%, and 1.0±0.6% for postmortem hearts with normal cardiac function (n=7), early-stage of mild hypertrophy (n=5), severe hypertrophy/dilation (n=4), and end-stage CHF (n=6), respectively. In fresh transplant samples, the %Ptotal of cTnI from non-failing donor (n=4), and end-stage failing hearts (n=10) were 49.5±5.9% and 18.8±2.9%, respectively. Top-down MS with electron capture dissociation unequivocally localized the altered phosphorylation sites to Ser22/23 and determined the order of phosphorylation/dephosphorylation. This study represents the first clinical application of top-down MS-based quantitative proteomics for biomarker discovery from tissues, highlighting the potential of PTM as disease biomarkers. PMID:21751783
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Red blood cell (RBC) membrane proteomics--Part I: Proteomics and RBC physiology.
Pasini, Erica M; Lutz, Hans U; Mann, Matthias; Thomas, Alan W
2010-01-03
Membrane proteomics is concerned with accurately and sensitively identifying molecules involved in cell compartmentalisation, including those controlling the interface between the cell and the outside world. The high lipid content of the environment in which these proteins are found often causes a particular set of problems that must be overcome when isolating the required material before effective HPLC-MS approaches can be performed. The membrane is an unusually dynamic cellular structure since it interacts with an ever changing environment. A full understanding of this critical cell component will ultimately require, in addition to proteomics, lipidomics, glycomics, interactomics and study of post-translational modifications. Devoid of nucleus and organelles in mammalian species other than camelids, and constantly in motion in the blood stream, red blood cells (RBCs) are the sole mammalian oxygen transporter. The fact that mature mammalian RBCs have no internal membrane-bound organelles, somewhat simplifies proteomics analysis of the plasma membrane and the fact that it has no nucleus disqualifies microarray based methods. Proteomics has the potential to provide a better understanding of this critical interface, and thereby assist in identifying new approaches to diseases. (c) 2009 Elsevier B.V. All rights reserved.
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Quantitative proteomic analysis of human breast epithelial cells with differential telomere length
DOE Office of Scientific and Technical Information (OSTI.GOV)
Yu, Li-Rong; Chan, King C.; Tahara, Hidetoshi
Telomeres play important functional roles in cell proliferation, cell cycle regulation, and genetic stability, in which telomere length is critical. In this study, quantitative proteome comparisons for the human breast epithelial cells with short and long telomeres (184-hTERT{sub L} vs. 184-hTERT{sub S} and 90P-hTERT{sub L} vs. 90P-hTERT{sub S}), resulting from transfection of the human telomerase reverse transcriptase (hTERT) gene, were performed using cleavable isotope-coded affinity tags. More than 2000 proteins were quantified in each comparative experiment, with approximately 77% of the proteins identified in both analyses. In the cells with long telomeres, significant and consistent alterations were observed in metabolismmore » (amino acid, nucleotide, and lipid metabolism), genetic information transmission (transcription and translation regulation, spliceosome and ribosome complexes), and cell signaling. Interestingly, the DNA excision repair pathway is enhanced, while integrin and its ligands are downregulated in the cells with long telomeres. These results may provide valuable information related to telomere functions.« less
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Gozal, David; Jortani, Saeed; Snow, Ayelet B.; Kheirandish-Gozal, Leila; Bhattacharjee, Rakesh; Kim, Jinkwan; Capdevila, Oscar Sans
2009-01-01
Rationale: Sleep studies are laborious, expensive, inaccessible, and inconvenient for diagnosing obstructive sleep apnea (OSA) in children. Objectives: To examine whether the urinary proteome uncovers specific clusters that are differentially expressed in the urine of children with OSA. Methods: Two-dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry proteomics followed by validation with western blot of ELISA. Measurements and Main Results: Morning urine proteins from 60 children with polysomnographically confirmed OSA and from matched children with primary snoring (n = 30) and control subjects (n = 30) were assessed. A total of 16 proteins that are differentially expressed in OSA were identified, and 7 were confirmed by either immunoblots or ELISA. Among the latter, receiver–operator curve analyses of urinary concentrations of uromodulin, urocortin-3, orosomucoid-1, and kallikrein assigned favorable predictive properties to these proteins. Furthermore, combinatorial approaches indicated that the presence of values beyond the calculated cutoff concentrations for three or more of the proteins yielded a sensitivity of 95% and a specificity of 100%. Conclusions: Proteomic approaches reveal that pediatric OSA is associated with specific and consistent alterations in urinary concentrations of specific protein clusters. Future studies aiming to validate this approach as a screening method of habitually snoring children appears warranted. PMID:19797158
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The quantitative proteomes of human-induced pluripotent stem cells and embryonic stem cells
Munoz, Javier; Low, Teck Y; Kok, Yee J; Chin, Angela; Frese, Christian K; Ding, Vanessa; Choo, Andre; Heck, Albert J R
2011-01-01
Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important, given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here, we present an in-depth quantitative mass spectrometry-based analysis of hESCs, two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless, a small group of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. PMID:22108792
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Proteomics of effector-triggered immunity (ETI) in plants.
Hurley, Brenden; Subramaniam, Rajagopal; Guttman, David S; Desveaux, Darrell
2014-01-01
Effector-triggered immunity (ETI) was originally termed gene-for-gene resistance and dates back to fundamental observations of flax resistance to rust fungi by Harold Henry Flor in the 1940s. Since then, genetic and biochemical approaches have defined our current understanding of how plant "resistance" proteins recognize microbial effectors. More recently, proteomic approaches have expanded our view of the protein landscape during ETI and contributed significant advances to our mechanistic understanding of ETI signaling. Here we provide an overview of proteomic techniques that have been used to study plant ETI including both global and targeted approaches. We discuss the challenges associated with ETI proteomics and highlight specific examples from the literature, which demonstrate how proteomics is advancing the ETI research field.
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Potential protein biomarkers for burning mouth syndrome discovered by quantitative proteomics
Ji, Eoon Hye; Diep, Cynthia; Liu, Tong; Li, Hong; Merrill, Robert; Messadi, Diana
2017-01-01
Burning mouth syndrome (BMS) is a chronic pain disorder characterized by severe burning sensation in normal looking oral mucosa. Diagnosis of BMS remains to be a challenge to oral healthcare professionals because the method for definite diagnosis is still uncertain. In this study, a quantitative saliva proteomic analysis was performed in order to identify target proteins in BMS patients’ saliva that may be used as biomarkers for simple, non-invasive detection of the disease. By using isobaric tags for relative and absolute quantitation labeling and liquid chromatography-tandem mass spectrometry to quantify 1130 saliva proteins between BMS patients and healthy control subjects, we found that 50 proteins were significantly changed in the BMS patients when compared to the healthy control subjects (p ≤ 0.05, 39 up-regulated and 11 down-regulated). Four candidates, alpha-enolase, interleukin-18 (IL-18), kallikrein-13 (KLK13), and cathepsin G, were selected for further validation. Based on enzyme-linked immunosorbent assay measurements, three potential biomarkers, alpha-enolase, IL-18, and KLK13, were successfully validated. The fold changes for alpha-enolase, IL-18, and KLK13 were determined as 3.6, 2.9, and 2.2 (burning mouth syndrome vs. control), and corresponding receiver operating characteristic values were determined as 0.78, 0.83, and 0.68, respectively. Our findings indicate that testing of the identified protein biomarkers in saliva might be a valuable clinical tool for BMS detection. Further validation studies of the identified biomarkers or additional candidate biomarkers are needed to achieve a multi-marker prediction model for improved detection of BMS with high sensitivity and specificity. PMID:28326926
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Potential protein biomarkers for burning mouth syndrome discovered by quantitative proteomics.
Ji, Eoon Hye; Diep, Cynthia; Liu, Tong; Li, Hong; Merrill, Robert; Messadi, Diana; Hu, Shen
2017-01-01
Burning mouth syndrome (BMS) is a chronic pain disorder characterized by severe burning sensation in normal looking oral mucosa. Diagnosis of BMS remains to be a challenge to oral healthcare professionals because the method for definite diagnosis is still uncertain. In this study, a quantitative saliva proteomic analysis was performed in order to identify target proteins in BMS patients' saliva that may be used as biomarkers for simple, non-invasive detection of the disease. By using isobaric tags for relative and absolute quantitation labeling and liquid chromatography-tandem mass spectrometry to quantify 1130 saliva proteins between BMS patients and healthy control subjects, we found that 50 proteins were significantly changed in the BMS patients when compared to the healthy control subjects ( p ≤ 0.05, 39 up-regulated and 11 down-regulated). Four candidates, alpha-enolase, interleukin-18 (IL-18), kallikrein-13 (KLK13), and cathepsin G, were selected for further validation. Based on enzyme-linked immunosorbent assay measurements, three potential biomarkers, alpha-enolase, IL-18, and KLK13, were successfully validated. The fold changes for alpha-enolase, IL-18, and KLK13 were determined as 3.6, 2.9, and 2.2 (burning mouth syndrome vs. control), and corresponding receiver operating characteristic values were determined as 0.78, 0.83, and 0.68, respectively. Our findings indicate that testing of the identified protein biomarkers in saliva might be a valuable clinical tool for BMS detection. Further validation studies of the identified biomarkers or additional candidate biomarkers are needed to achieve a multi-marker prediction model for improved detection of BMS with high sensitivity and specificity.
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Yang, Qiao-Song; Wu, Jun-Hua; Li, Chun-Yu; Wei, Yue-Rong; Sheng, Ou; Hu, Chun-Hua; Kuang, Rui-Bin; Huang, Yong-Hong; Peng, Xin-Xiang; McCardle, James A; Chen, Wei; Yang, Yong; Rose, Jocelyn K C; Zhang, Sheng; Yi, Gan-Jun
2012-12-01
Banana and its close relative, plantain are globally important crops and there is considerable interest in optimizing their cultivation. Plantain has superior cold tolerance compared with banana and a thorough understanding of the molecular mechanisms and responses of plantain to cold stress has great potential value for developing cold tolerant banana cultivars. In this study, we used iTRAQ-based comparative proteomic analysis to investigate the temporal responses of plantain to cold stress. Plantain seedlings were exposed for 0, 6, and 24 h of cold stress at 8 °C and subsequently allowed to recover for 24 h at 28 °C. A total of 3477 plantain proteins were identified, of which 809 showed differential expression from the three treatments. The majority of differentially expressed proteins were predicted to be involved in oxidation-reduction, including oxylipin biosynthesis, whereas others were associated with photosynthesis, photorespiration, and several primary metabolic processes, such as carbohydrate metabolic process and fatty acid beta-oxidation. Western blot analysis and enzyme activity assays were performed on seven differentially expressed, cold-response candidate plantain proteins to validate the proteomics data. Similar analyses of the seven candidate proteins were performed in cold-sensitive banana to examine possible functional conservation, and to compare the results to equivalent responses between the two species. Consistent results were achieved by Western blot and enzyme activity assays, demonstrating that the quantitative proteomics data collected in this study are reliable. Our results suggest that an increase of antioxidant capacity through adapted ROS scavenging capability, reduced production of ROS, and decreased lipid peroxidation contribute to molecular mechanisms for the increased cold tolerance in plantain. To the best of our knowledge, this is the first report of a global investigation on molecular responses of plantain to cold stress by
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Yang, Qiao-Song; Wu, Jun-Hua; Li, Chun-Yu; Wei, Yue-Rong; Sheng, Ou; Hu, Chun-Hua; Kuang, Rui-Bin; Huang, Yong-Hong; Peng, Xin-Xiang; McCardle, James A.; Chen, Wei; Yang, Yong; Rose, Jocelyn K. C.; Zhang, Sheng; Yi, Gan-Jun
2012-01-01
Banana and its close relative, plantain are globally important crops and there is considerable interest in optimizing their cultivation. Plantain has superior cold tolerance compared with banana and a thorough understanding of the molecular mechanisms and responses of plantain to cold stress has great potential value for developing cold tolerant banana cultivars. In this study, we used iTRAQ-based comparative proteomic analysis to investigate the temporal responses of plantain to cold stress. Plantain seedlings were exposed for 0, 6, and 24 h of cold stress at 8 °C and subsequently allowed to recover for 24 h at 28 °C. A total of 3477 plantain proteins were identified, of which 809 showed differential expression from the three treatments. The majority of differentially expressed proteins were predicted to be involved in oxidation-reduction, including oxylipin biosynthesis, whereas others were associated with photosynthesis, photorespiration, and several primary metabolic processes, such as carbohydrate metabolic process and fatty acid beta-oxidation. Western blot analysis and enzyme activity assays were performed on seven differentially expressed, cold-response candidate plantain proteins to validate the proteomics data. Similar analyses of the seven candidate proteins were performed in cold-sensitive banana to examine possible functional conservation, and to compare the results to equivalent responses between the two species. Consistent results were achieved by Western blot and enzyme activity assays, demonstrating that the quantitative proteomics data collected in this study are reliable. Our results suggest that an increase of antioxidant capacity through adapted ROS scavenging capability, reduced production of ROS, and decreased lipid peroxidation contribute to molecular mechanisms for the increased cold tolerance in plantain. To the best of our knowledge, this is the first report of a global investigation on molecular responses of plantain to cold stress by
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Megger, Dominik A.; Philipp, Jos; Le-Trilling, Vu Thuy Khanh; Sitek, Barbara; Trilling, Mirko
2017-01-01
Interferons (IFNs) are pleotropic cytokines secreted upon encounter of pathogens and tumors. Applying their antipathogenic, antiproliferative, and immune stimulatory capacities, recombinant IFNs are frequently prescribed as drugs to treat different diseases. IFNs act by changing the gene expression profile of cells. Due to characteristics such as rapid gene induction and signaling, IFNs also represent prototypical model systems for various aspects of biomedical research (e.g., signal transduction). In regard to the signaling and activated promoters, IFNs can be subdivided into two groups. Here, alterations of the cellular proteome of human cells treated with IFNα and IFNγ were elucidated in a time-resolved manner by quantitative proteome analysis. The majority of protein regulations were strongly IFN type and time dependent. In addition to the expected upregulation of IFN-responsive proteins, an astonishing number of proteins became profoundly repressed especially by IFNγ. Thus, our comprehensive analysis revealed important insights into the human IFN-regulated proteome and its dynamics of protein induction and repression. Interestingly, the new class of IFN-repressed genes comprises known host factors for highly relevant pathogens such as HIV, dengue virus, and hepatitis C virus. PMID:28959263
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Megger, Dominik A; Philipp, Jos; Le-Trilling, Vu Thuy Khanh; Sitek, Barbara; Trilling, Mirko
2017-01-01
Interferons (IFNs) are pleotropic cytokines secreted upon encounter of pathogens and tumors. Applying their antipathogenic, antiproliferative, and immune stimulatory capacities, recombinant IFNs are frequently prescribed as drugs to treat different diseases. IFNs act by changing the gene expression profile of cells. Due to characteristics such as rapid gene induction and signaling, IFNs also represent prototypical model systems for various aspects of biomedical research (e.g., signal transduction). In regard to the signaling and activated promoters, IFNs can be subdivided into two groups. Here, alterations of the cellular proteome of human cells treated with IFNα and IFNγ were elucidated in a time-resolved manner by quantitative proteome analysis. The majority of protein regulations were strongly IFN type and time dependent. In addition to the expected upregulation of IFN-responsive proteins, an astonishing number of proteins became profoundly repressed especially by IFNγ. Thus, our comprehensive analysis revealed important insights into the human IFN-regulated proteome and its dynamics of protein induction and repression. Interestingly, the new class of IFN-repressed genes comprises known host factors for highly relevant pathogens such as HIV, dengue virus, and hepatitis C virus.
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A multicenter study benchmarks software tools for label-free proteome quantification.
Navarro, Pedro; Kuharev, Jörg; Gillet, Ludovic C; Bernhardt, Oliver M; MacLean, Brendan; Röst, Hannes L; Tate, Stephen A; Tsou, Chih-Chiang; Reiter, Lukas; Distler, Ute; Rosenberger, George; Perez-Riverol, Yasset; Nesvizhskii, Alexey I; Aebersold, Ruedi; Tenzer, Stefan
2016-11-01
Consistent and accurate quantification of proteins by mass spectrometry (MS)-based proteomics depends on the performance of instruments, acquisition methods and data analysis software. In collaboration with the software developers, we evaluated OpenSWATH, SWATH 2.0, Skyline, Spectronaut and DIA-Umpire, five of the most widely used software methods for processing data from sequential window acquisition of all theoretical fragment-ion spectra (SWATH)-MS, which uses data-independent acquisition (DIA) for label-free protein quantification. We analyzed high-complexity test data sets from hybrid proteome samples of defined quantitative composition acquired on two different MS instruments using different SWATH isolation-window setups. For consistent evaluation, we developed LFQbench, an R package, to calculate metrics of precision and accuracy in label-free quantitative MS and report the identification performance, robustness and specificity of each software tool. Our reference data sets enabled developers to improve their software tools. After optimization, all tools provided highly convergent identification and reliable quantification performance, underscoring their robustness for label-free quantitative proteomics.
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Han, Bomie; Higgs, Richard E
2008-09-01
High-throughput HPLC-mass spectrometry (HPLC-MS) is routinely used to profile biological samples for potential protein markers of disease, drug efficacy and toxicity. The discovery technology has advanced to the point where translating hypotheses from proteomic profiling studies into clinical use is the bottleneck to realizing the full potential of these approaches. The first step in this translation is the development and analytical validation of a higher throughput assay with improved sensitivity and selectivity relative to typical profiling assays. Multiple reaction monitoring (MRM) assays are an attractive approach for this stage of biomarker development given their improved sensitivity and specificity, the speed at which the assays can be developed and the quantitative nature of the assay. While the profiling assays are performed with ion trap mass spectrometers, MRM assays are traditionally developed in quadrupole-based mass spectrometers. Development of MRM assays from the same instrument used in the profiling analysis enables a seamless and rapid transition from hypothesis generation to validation. This report provides guidelines for rapidly developing an MRM assay using the same mass spectrometry platform used for profiling experiments (typically ion traps) and reviews methodological and analytical validation considerations. The analytical validation guidelines presented are drawn from existing practices on immunological assays and are applicable to any mass spectrometry platform technology.
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Chen, Xiulan; Wei, Shasha; Ma, Ying; Lu, Jie; Niu, Gang; Xue, Yanhong; Chen, Xiaoyuan; Yang, Fuquan
2014-01-01
Doxorubicin is a widely used chemotherapeutic agent for the treatment of a variety of solid tumors. However, resistance to this anticancer drug is a major obstacle to the effective treatment of tumors. As mitochondria play important roles in cell life and death, we anticipate that mitochondria may be related to drug resistance. Here, stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic strategy was applied to compare mitochondrial protein expression in doxorubicin sensitive OVCAR8 cells and its doxorubicin-resistant variant NCI_ADR/RES cells. A total of 2085 proteins were quantified, of which 122 proteins displayed significant changes in the NCI_ADR/RES cells. These proteins participated in a variety of cell processes including cell apoptosis, substance metabolism, transport, detoxification and drug metabolism. Then qRT-PCR and western blot were applied to validate the differentially expressed proteins quantified by SILAC. Further functional studies with RNAi demonstrated TOP1MT, a mitochondrial protein participated in DNA repair, was involved in doxorubicin resistance in NCI_ADR/RES cells. Besides the proteomic study, electron microscopy and fluorescence analysis also observed that mitochondrial morphology and localization were greatly altered in NCI_ADR/RES cells. Mitochondrial membrane potential was also decreased in NCI_ADR/RES cells. All these results indicate that mitochondrial function is impaired in doxorubicin-resistant cells and mitochondria play an important role in doxorubicin resistance. This research provides some new information about doxorubicin resistance, indicating that mitochondria could be therapeutic targets of doxorubicin resistance in ovarian cancer cells. PMID:25285166
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Computer aided manual validation of mass spectrometry-based proteomic data.
Curran, Timothy G; Bryson, Bryan D; Reigelhaupt, Michael; Johnson, Hannah; White, Forest M
2013-06-15
Advances in mass spectrometry-based proteomic technologies have increased the speed of analysis and the depth provided by a single analysis. Computational tools to evaluate the accuracy of peptide identifications from these high-throughput analyses have not kept pace with technological advances; currently the most common quality evaluation methods are based on statistical analysis of the likelihood of false positive identifications in large-scale data sets. While helpful, these calculations do not consider the accuracy of each identification, thus creating a precarious situation for biologists relying on the data to inform experimental design. Manual validation is the gold standard approach to confirm accuracy of database identifications, but is extremely time-intensive. To palliate the increasing time required to manually validate large proteomic datasets, we provide computer aided manual validation software (CAMV) to expedite the process. Relevant spectra are collected, catalogued, and pre-labeled, allowing users to efficiently judge the quality of each identification and summarize applicable quantitative information. CAMV significantly reduces the burden associated with manual validation and will hopefully encourage broader adoption of manual validation in mass spectrometry-based proteomics. Copyright © 2013 Elsevier Inc. All rights reserved.
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Sun, Huishan; Pan, Liping; Jia, Hongyan; Zhang, Zhiguo; Gao, Mengqiu; Huang, Mailing; Wang, Jinghui; Sun, Qi; Wei, Rongrong; Du, Boping; Xing, Aiying; Zhang, Zongde
2018-01-01
The lack of effective differential diagnostic methods for active tuberculosis (TB) and latent infection (LTBI) is still an obstacle for TB control. Furthermore, the molecular mechanism behind the progression from LTBI to active TB has been not elucidated. Therefore, we performed label-free quantitative proteomics to identify plasma biomarkers for discriminating pulmonary TB (PTB) from LTBI. A total of 31 overlapping proteins with significant difference in expression level were identified in PTB patients ( n = 15), compared with LTBI individuals ( n = 15) and healthy controls (HCs, n = 15). Eight differentially expressed proteins were verified using western blot analysis, which was 100% consistent with the proteomics results. Statistically significant differences of six proteins were further validated in the PTB group compared with the LTBI and HC groups in the training set ( n = 240), using ELISA. Classification and regression tree (CART) analysis was employed to determine the ideal protein combination for discriminating PTB from LTBI and HC. A diagnostic model consisting of alpha-1-antichymotrypsin (ACT), alpha-1-acid glycoprotein 1 (AGP1), and E-cadherin (CDH1) was established and presented a sensitivity of 81.2% (69/85) and a specificity of 95.2% (80/84) in discriminating PTB from LTBI, and a sensitivity of 81.2% (69/85) and a specificity of 90.1% (64/81) in discriminating PTB from HCs. Additional validation was performed by evaluating the diagnostic model in blind testing set ( n = 113), which yielded a sensitivity of 75.0% (21/28) and specificity of 96.1% (25/26) in PTB vs. LTBI, 75.0% (21/28) and 92.3% (24/26) in PTB vs. HCs, and 75.0% (21/28) and 81.8% (27/33) in PTB vs. lung cancer (LC), respectively. This study obtained the plasma proteomic profiles of different M.TB infection statuses, which contribute to a better understanding of the pathogenesis involved in the transition from latent infection to TB activation and provide new potential diagnostic
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Zakirova, Zuchra; Reed, Jon; Crynen, Gogce; Horne, Lauren; Hassan, Samira; Mathura, Venkatarajan; Mullan, Michael; Crawford, Fiona; Ait-Ghezala, Ghania
2017-09-01
Long-term consequences of combined pyridostigmine bromide (PB) and permethrin (PER) exposure in C57BL6/J mice using a well-characterized mouse model of exposure to these Gulf War (GW) agents were explored at the protein level. We used orthogonal proteomic approaches to identify pathways that are chronically impacted in the mouse CNS due to semiacute GW agent exposure early in life. These analyses were performed on soluble and membrane-bound protein fractions from brain samples using two orthogonal isotopic labeling LC-MS/MS proteomic approaches-stable isotope dimethyl labeling and iTRAQ. The use of these approaches allowed for greater coverage of proteins than was possible by either one alone and revealed both distinct and overlapping datasets. This combined analysis identified changes in several mitochondrial, as well as immune and inflammatory pathways after GW agent exposure. The work discussed here provides insight into GW agent exposure dependent mechanisms that adversely affect mitochondrial function and immune and inflammatory regulation. Collectively, our work identified key pathways which were chronically impacted in the mouse CNS following acute GW agent exposure, this may lead to the identification of potential targets for therapeutic intervention in the future. Long-term consequences of combined PB and PER exposure in C57BL6/J mice using a well-characterized mouse model of exposure to these GW agents were explored at the protein level. Expanding on earlier work, we used orthogonal proteomic approaches to identify pathways that are chronically impacted in the mouse CNS due to semiacute GW agent exposure early in life. These analyses were performed on soluble and membrane-bound protein fractions from brain samples using two orthogonal isotopic labeling LC-MS/MS proteomic approaches-stable isotope dimethyl labeling and iTRAQ. The use of these approaches allowed for greater coverage of proteins than was possible by either one alone and revealed both
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Aasebø, Elise; Forthun, Rakel B.; Berven, Frode; Selheim, Frode; Hernandez-Valladares, Maria
2016-01-01
The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applications in AML diagnosis and prognosis, treatment and the selection for bone marrow transplant requires substantial comparative analyses of the proteomes from AML patients. In the past years, several studies have suggested some biomarkers for AML diagnosis or AML classification using methods for sample preparation with low proteome coverage and low resolution mass spectrometers. However, most of the studies did not follow up, confirm or validate their candidates with more patient samples. Current proteomics methods, new high resolution and fast mass spectrometers allow the identification and quantification of several thousands of proteins obtained from few tens of μg of AML cell lysate. Enrichment methods for posttranslational modifications (PTM), such as phosphorylation, can isolate several thousands of site-specific phosphorylated peptides from AML patient samples, which subsequently can be quantified with high confidence in new mass spectrometers. While recent reports aiming to propose proteomic or phosphoproteomic biomarkers on the studied AML patient samples have taken advantage of the technological progress, the access to large cohorts of AML patients to sample from and the availability of appropriate control samples still remain challenging. PMID:26306748
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Rokyta, Darin R; Ward, Micaiah J
2017-03-15
The order Scorpiones is one of the most ancient and diverse lineages of venomous animals, having originated approximately 430 million years ago and diversified into 14 extant families. Although partial venom characterizations have been described for numerous scorpion species, we provided the first quantitative transcriptome/proteome comparison for a scorpion species using single-animal approaches. We sequenced the venom-gland transcriptomes of a male and female black-back scorpion (Hadrurus spadix) from the family Caraboctonidae using the Illumina sequencing platform and conducted independent quantitative mass-spectrometry analyses of their venoms. We identified 79 proteomically confirmed venom proteins, an additional 69 transcripts with homology to toxins from other species, and 596 nontoxin proteins expressed at high levels in the venom glands. The venom of H. spadix was rich in antimicrobial peptides, K + -channel toxins, and several classes of peptidases. However, the most diverse and one of the most abundant classes of putative toxins could not be assigned even a tentative functional role on the basis of homology, indicating that this venom contained a wealth of previously unexplored animal toxin diversity. We found good agreement between both transcriptomic and proteomic abundances across individuals, but transcriptomic and proteomic abundandances differed substantially within each individual. Small peptide toxins such as K + -channel toxins and antimicrobial peptides proved challenging to detect proteomically, at least in part due to the significant proteolytic processing involved in their maturation. In addition, we found a significant tendency for our proteomic approach to overestimate the abundances of large putative toxins and underestimate the abundances of smaller toxins. Copyright © 2017 Elsevier Ltd. All rights reserved.
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MASH Suite Pro: A Comprehensive Software Tool for Top-Down Proteomics*
Cai, Wenxuan; Guner, Huseyin; Gregorich, Zachery R.; Chen, Albert J.; Ayaz-Guner, Serife; Peng, Ying; Valeja, Santosh G.; Liu, Xiaowen; Ge, Ying
2016-01-01
Top-down mass spectrometry (MS)-based proteomics is arguably a disruptive technology for the comprehensive analysis of all proteoforms arising from genetic variation, alternative splicing, and posttranslational modifications (PTMs). However, the complexity of top-down high-resolution mass spectra presents a significant challenge for data analysis. In contrast to the well-developed software packages available for data analysis in bottom-up proteomics, the data analysis tools in top-down proteomics remain underdeveloped. Moreover, despite recent efforts to develop algorithms and tools for the deconvolution of top-down high-resolution mass spectra and the identification of proteins from complex mixtures, a multifunctional software platform, which allows for the identification, quantitation, and characterization of proteoforms with visual validation, is still lacking. Herein, we have developed MASH Suite Pro, a comprehensive software tool for top-down proteomics with multifaceted functionality. MASH Suite Pro is capable of processing high-resolution MS and tandem MS (MS/MS) data using two deconvolution algorithms to optimize protein identification results. In addition, MASH Suite Pro allows for the characterization of PTMs and sequence variations, as well as the relative quantitation of multiple proteoforms in different experimental conditions. The program also provides visualization components for validation and correction of the computational outputs. Furthermore, MASH Suite Pro facilitates data reporting and presentation via direct output of the graphics. Thus, MASH Suite Pro significantly simplifies and speeds up the interpretation of high-resolution top-down proteomics data by integrating tools for protein identification, quantitation, characterization, and visual validation into a customizable and user-friendly interface. We envision that MASH Suite Pro will play an integral role in advancing the burgeoning field of top-down proteomics. PMID:26598644
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Xia, Kai; Zang, Ning; Zhang, Junmei; Zhang, Hong; Li, Yudong; Liu, Ye; Feng, Wei; Liang, Xinle
2016-12-05
Acetobacter pasteurianus is the main starter in rice vinegar manufacturing due to its remarkable abilities to resist and produce acetic acid. Although several mechanisms of acetic acid resistance have been proposed and only a few effector proteins have been identified, a comprehensive depiction of the biological processes involved in acetic acid resistance is needed. In this study, iTRAQ-based quantitative proteomic analysis was adopted to investigate the whole proteome of different acidic titers (3.6, 7.1 and 9.3%, w/v) of Acetobacter pasteurianus Ab3 during the vinegar fermentation process. Consequently, 1386 proteins, including 318 differentially expressed proteins (p<0.05), were identified. Compared to that in the low titer circumstance, cells conducted distinct biological processes under high acetic acid stress, where >150 proteins were differentially expressed. Specifically, proteins involved in amino acid metabolic processes and fatty acid biosynthesis were differentially expressed, which may contribute to the acetic acid resistance of Acetobacter. Transcription factors, two component systems and toxin-antitoxin systems were implicated in the modulatory network at multiple levels. In addition, the identification of proteins involved in redox homeostasis, protein metabolism, and the cell envelope suggested that the whole cellular system is mobilized in response to acid stress. These findings provide a differential proteomic profile of acetic acid resistance in Acetobacter pasteurianus and have potential application to highly acidic rice vinegar manufacturing. Copyright © 2016 Elsevier B.V. All rights reserved.
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Zhang, Liyuan; Yu, Zhenlong; Wang, Yan; Wang, Xiaobo; Zhang, Lianru; Wang, Chao; Yue, Qingxi; Wang, Xun; Deng, Sa; Huo, Xiaokui; Tian, Xiangge; Huang, Shanshan; Zhang, Baojing; Ma, Xiaochi
2016-11-22
Gamabufotalin (CS-6) is a major bufadienolide of Chansu, which shows desirable metabolic stability and less adverse effect in cancer therapy. CS-6 treatment inhibited the proliferation of NSCLC in a nanomolar range. And CS-6 could induce G2/M cell cycle arrest and apoptosis in A549 cells. However, its molecular mechanism in antitumor activity remains poorly understood. We employed a quantitative proteomics approach to identify the potential cellular targets of CS-6, and found 38 possible target-related proteins. Among them, 31 proteins were closely related in the protein-protein interaction network. One of the regulatory nodes in key pathways was occupied by Hsp90. Molecular docking revealed that CS-6 interacted with the ATP-binding sites of Hsp90. In addition, CS-6 inhibited the chaperone function of Hsp90 and reduced expression of Hsp90-dependent client proteins. Moreover, CS-6 markedly down-regulated the protein level of Hsp90 in tumor tissues of the xenograft mice. Taken together, our results suggest that CS-6 might be a novel inhibitor of Hsp90, and the possible network associated with CS-6 target-related proteins was constructed, which provided experimental evidence for the preclinical value of using CS-6 as an effective antitumor agent in treatment of NSCLC.
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Tu, Chengjian; Mojica, Wilfrido; Straubinger, Robert M; Li, Jun; Shen, Shichen; Qu, Miao; Nie, Lei; Roberts, Rick; An, Bo; Qu, Jun
2017-05-01
The heterogeneous structure in tumor tissues from colorectal cancer (CRC) patients excludes an informative comparison between tumors and adjacent normal tissues. Here, we develop and apply a strategy to compare paired cancerous (CEC) versus normal (NEC) epithelial cells enriched from patients and discover potential biomarkers and therapeutic targets for CRC. CEC and NEC cells are respectively isolated from five different tumor and normal locations in the resected colon tissue from each patient (N = 12 patients) using an optimized epithelial cell adhesion molecule (EpCAM)-based enrichment approach. An ion current-based quantitative method is employed to perform comparative proteomic analysis for each patient. A total of 458 altered proteins that are common among >75% of patients are observed and selected for further investigation. Besides known findings such as deregulation of mitochondrial function, tricarboxylic acid cycle, and RNA post-transcriptional modification, functional analysis further revealed RAN signaling pathway, small nucleolar ribonucleoproteins (snoRNPs), and infection by RNA viruses are altered in CEC cells. A selection of the altered proteins of interest is validated by immunohistochemistry analyses. The informative comparison between matched CEC and NEC enhances our understanding of molecular mechanisms of CRC development and provides biomarker candidates and new pathways for therapeutic intervention. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Proteomics for understanding miRNA biology.
Huang, Tai-Chung; Pinto, Sneha M; Pandey, Akhilesh
2013-02-01
MicroRNAs (miRNAs) are small noncoding RNAs that play important roles in posttranscriptional regulation of gene expression. Mature miRNAs associate with the RNA interference silencing complex to repress mRNA translation and/or degrade mRNA transcripts. Mass spectrometry-based proteomics has enabled identification of several core components of the canonical miRNA processing pathway and their posttranslational modifications which are pivotal in miRNA regulatory mechanisms. The use of quantitative proteomic strategies has also emerged as a key technique for experimental identification of miRNA targets by allowing direct determination of proteins whose levels are altered because of translational suppression. This review focuses on the role of proteomics and labeling strategies to understand miRNA biology. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Marcon, Caroline; Lamkemeyer, Tobias; Malik, Waqas Ahmed; Ungrue, Denise; Piepho, Hans-Peter; Hochholdinger, Frank
2013-11-20
Heterosis is the superior performance of heterozygous F1-hybrid plants compared to their homozygous genetically distinct parents. Seminal roots are embryonic roots that play an important role during early maize (Zea mays L.) seedling development. In the present study the most abundant soluble proteins of 2-4cm seminal roots of the reciprocal maize F1-hybrids B73×Mo17 and Mo17×B73 and their parental inbred lines B73 and Mo17 were quantified by label-free LC-MS/MS. In total, 1918 proteins were detected by this shot-gun approach. Among those, 970 were represented by at least two peptides and were further analyzed. Eighty-five proteins displayed non-additive accumulation in at least one hybrid. The functional category protein metabolism was the most abundant class of non-additive proteins represented by 27 proteins. Within this category 16 of 17 non-additively accumulated ribosomal proteins showed high or above high parent expression in seminal roots. These results imply that an increased protein synthesis rate in hybrids might be related to the early manifestation of hybrid vigor in seminal roots. In the present study a shot-gun proteomics approach allowed for the identification of 1917 proteins and analysis of 970 seminal root proteins of maize that were represented by at least 2 peptides. The comparison of proteome complexity of reciprocal hybrids and their parental inbred lines indicates an increased protein synthesis rate in hybrids that may contribute to the early manifestation of heterosis in seminal roots. This article is part of a Special Issue entitled: Translational Plant Proteomics. Copyright © 2013 Elsevier B.V. All rights reserved.
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A proteomic approach to understanding the pathogenesis of idiopathic macular hole formation.
Zhang, Pingbo; Zhu, Min; Zhao, Yuming; Qian, Jiang; Dufresne, Craig; Turner, Randi; Semba, Richard D; Solomon, Sharon D
2017-01-01
Idiopathic macular holes (IMH) are full-thickness defects of retinal tissue that cause severe vision loss due to disruption of the anatomic fovea. Abnormal vitreous traction is involved in the formation of macular holes. Both glial cells and hyalocytes contribute to epiretinal membrane formation in IMH. In order to gain further insight into the pathophysiology of IMH, we conducted a discovery phase investigation of the vitreous proteome in four patients with macular holes and six controls using one-dimensional gel fractionation and liquid chromatography-tandem mass spectrometry analyses on an Orbitrap Elite mass spectrometer. Of a total of 5912 vitreous proteins, 32 proteins had increased and 39 proteins had decreased expression in IMH compared with controls, using a false discovery rate approach with p value < 0.001 and q value < 0.05. IMH was associated with increased expression of proteins in the complement pathway, α-2-macroglobulin, a major inducer of Müller glial cell migration, fibrinogen, and extracellular matrix proteins, and decreased expression of proteins involved in protein folding and actin filament binding. A proteomic approach revealed proteins and biological pathways that may be involved in the pathogenesis of IMH and could be targeted for future studies.
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Principles of proteome allocation are revealed using proteomic data and genome-scale models
Yang, Laurence; Yurkovich, James T.; Lloyd, Colton J.; Ebrahim, Ali; Saunders, Michael A.; Palsson, Bernhard O.
2016-01-01
Integrating omics data to refine or make context-specific models is an active field of constraint-based modeling. Proteomics now cover over 95% of the Escherichia coli proteome by mass. Genome-scale models of Metabolism and macromolecular Expression (ME) compute proteome allocation linked to metabolism and fitness. Using proteomics data, we formulated allocation constraints for key proteome sectors in the ME model. The resulting calibrated model effectively computed the “generalist” (wild-type) E. coli proteome and phenotype across diverse growth environments. Across 15 growth conditions, prediction errors for growth rate and metabolic fluxes were 69% and 14% lower, respectively. The sector-constrained ME model thus represents a generalist ME model reflecting both growth rate maximization and “hedging” against uncertain environments and stresses, as indicated by significant enrichment of these sectors for the general stress response sigma factor σS. Finally, the sector constraints represent a general formalism for integrating omics data from any experimental condition into constraint-based ME models. The constraints can be fine-grained (individual proteins) or coarse-grained (functionally-related protein groups) as demonstrated here. This flexible formalism provides an accessible approach for narrowing the gap between the complexity captured by omics data and governing principles of proteome allocation described by systems-level models. PMID:27857205
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Principles of proteome allocation are revealed using proteomic data and genome-scale models
Yang, Laurence; Yurkovich, James T.; Lloyd, Colton J.; ...
2016-11-18
Integrating omics data to refine or make context-specific models is an active field of constraint-based modeling. Proteomics now cover over 95% of the Escherichia coli proteome by mass. Genome-scale models of Metabolism and macromolecular Expression (ME) compute proteome allocation linked to metabolism and fitness. Using proteomics data, we formulated allocation constraints for key proteome sectors in the ME model. The resulting calibrated model effectively computed the “generalist” (wild-type) E. coli proteome and phenotype across diverse growth environments. Across 15 growth conditions, prediction errors for growth rate and metabolic fluxes were 69% and 14% lower, respectively. The sector-constrained ME model thusmore » represents a generalist ME model reflecting both growth rate maximization and “hedging” against uncertain environments and stresses, as indicated by significant enrichment of these sectors for the general stress response sigma factor σS. Finally, the sector constraints represent a general formalism for integrating omics data from any experimental condition into constraint-based ME models. The constraints can be fine-grained (individual proteins) or coarse-grained (functionally-related protein groups) as demonstrated here. Furthermore, this flexible formalism provides an accessible approach for narrowing the gap between the complexity captured by omics data and governing principles of proteome allocation described by systems-level models.« less
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An overview of quantitative approaches in Gestalt perception.
Jäkel, Frank; Singh, Manish; Wichmann, Felix A; Herzog, Michael H
2016-09-01
Gestalt psychology is often criticized as lacking quantitative measurements and precise mathematical models. While this is true of the early Gestalt school, today there are many quantitative approaches in Gestalt perception and the special issue of Vision Research "Quantitative Approaches in Gestalt Perception" showcases the current state-of-the-art. In this article we give an overview of these current approaches. For example, ideal observer models are one of the standard quantitative tools in vision research and there is a clear trend to try and apply this tool to Gestalt perception and thereby integrate Gestalt perception into mainstream vision research. More generally, Bayesian models, long popular in other areas of vision research, are increasingly being employed to model perceptual grouping as well. Thus, although experimental and theoretical approaches to Gestalt perception remain quite diverse, we are hopeful that these quantitative trends will pave the way for a unified theory. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Barkla, Bronwyn J; Vera-Estrella, Rosario; Raymond, Carolyn
2016-05-10
Epidermal bladder cells (EBC) are large single-celled, specialized, and modified trichomes found on the aerial parts of the halophyte Mesembryanthemum crystallinum. Recent development of a simple but high throughput technique to extract the contents from these cells has provided an opportunity to conduct detailed single-cell-type analyses of their molecular characteristics at high resolution to gain insight into the role of these cells in the salt tolerance of the plant. In this study, we carry out large-scale complementary quantitative proteomic studies using both a label (DIGE) and label-free (GeLC-MS) approach to identify salt-responsive proteins in the EBC extract. Additionally we perform an ionomics analysis (ICP-MS) to follow changes in the amounts of 27 different elements. Using these methods, we were able to identify 54 proteins and nine elements that showed statistically significant changes in the EBC from salt-treated plants. GO enrichment analysis identified a large number of transport proteins but also proteins involved in photosynthesis, primary metabolism and Crassulacean acid metabolism (CAM). Validation of results by western blot, confocal microscopy and enzyme analysis helped to strengthen findings and further our understanding into the role of these specialized cells. As expected EBC accumulated large quantities of sodium, however, the most abundant element was chloride suggesting the sequestration of this ion into the EBC vacuole is just as important for salt tolerance. This single-cell type omics approach shows that epidermal bladder cells of M. crystallinum are metabolically active modified trichomes, with primary metabolism supporting cell growth, ion accumulation, compatible solute synthesis and CAM. Data are available via ProteomeXchange with identifier PXD004045.
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Casey, Tammy M; Khan, Javed M; Bringans, Scott D; Koudelka, Tomas; Takle, Pari S; Downs, Rachael A; Livk, Andreja; Syme, Robert A; Tan, Kar-Chun; Lipscombe, Richard J
2017-02-03
This study aimed to compare the depth and reproducibility of total proteome and differentially expressed protein coverage in technical duplicates and triplicates using iTRAQ 4-plex, iTRAQ 8-plex, and TMT 6-plex reagents. The analysis was undertaken because comprehensive comparisons of isobaric mass tag reproducibility have not been widely reported in the literature. The highest number of proteins was identified with 4-plex, followed by 8-plex and then 6-plex reagents. Quantitative analyses revealed that more differentially expressed proteins were identified with 4-plex reagents than 8-plex reagents and 6-plex reagents. Replicate reproducibility was determined to be ≥69% for technical duplicates and ≥57% for technical triplicates. The results indicate that running an 8-plex or 6-plex experiment instead of a 4-plex experiment resulted in 26 or 39% fewer protein identifications, respectively. When 4-plex spectra were searched with three software tools-ProteinPilot, Mascot, and Proteome Discoverer-the highest number of protein identifications were obtained with Mascot. The analysis of negative controls demonstrated the importance of running experiments as replicates. Overall, this study demonstrates the advantages of using iTRAQ 4-plex reagents over iTRAQ 8-plex and TMT 6-plex reagents, provides estimates of technical duplicate and triplicate reproducibility, and emphasizes the value of running replicate samples.
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The wheat chloroplastic proteome.
Kamal, Abu Hena Mostafa; Cho, Kun; Choi, Jong-Soon; Bae, Kwang-Hee; Komatsu, Setsuko; Uozumi, Nobuyuki; Woo, Sun Hee
2013-11-20
With the availability of plant genome sequencing, analysis of plant proteins with mass spectrometry has become promising and admired. Determining the proteome of a cell is still a challenging assignment, which is convoluted by proteome dynamics and convolution. Chloroplast is fastidious curiosity for plant biologists due to their intricate biochemical pathways for indispensable metabolite functions. In this review, an overview on proteomic studies conducted in wheat with a special focus on subcellular proteomics of chloroplast, salt and water stress. In recent years, we and other groups have attempted to understand the photosynthesis in wheat and abiotic stress under salt imposed and water deficit during vegetative stage. Those studies provide interesting results leading to better understanding of the photosynthesis and identifying the stress-responsive proteins. Indeed, recent studies aimed at resolving the photosynthesis pathway in wheat. Proteomic analysis combining two complementary approaches such as 2-DE and shotgun methods couple to high through put mass spectrometry (LTQ-FTICR and MALDI-TOF/TOF) in order to better understand the responsible proteins in photosynthesis and abiotic stress (salt and water) in wheat chloroplast will be focused. In this review we discussed the identification of the most abundant protein in wheat chloroplast and stress-responsive under salt and water stress in chloroplast of wheat seedlings, thus providing the proteomic view of the events during the development of this seedling under stress conditions. Chloroplast is fastidious curiosity for plant biologists due to their intricate biochemical pathways for indispensable metabolite functions. An overview on proteomic studies conducted in wheat with a special focus on subcellular proteomics of chloroplast, salt and water stress. We have attempted to understand the photosynthesis in wheat and abiotic stress under salt imposed and water deficit during seedling stage. Those studies
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Lee, Jiyeong; Joo, Eun-Jeong; Lim, Hee-Joung; Park, Jong-Moon; Lee, Kyu Young; Park, Arum; Seok, AeEun
2015-01-01
Objective Currently, there are a few biological markers to aid in the diagnosis and treatment of depression. However, it is not sufficient for diagnosis. We attempted to identify differentially expressed proteins during depressive moods as putative diagnostic biomarkers by using quantitative proteomic analysis of serum. Methods Blood samples were collected twice from five patients with major depressive disorder (MDD) at depressive status before treatment and at remission status during treatment. Samples were individually analyzed by liquid chromatography-tandem mass spectrometry for protein profiling. Differentially expressed proteins were analyzed by label-free quantification. Enzyme-linked immunosorbent assay (ELISA) results and receiver-operating characteristic (ROC) curves were used to validate the differentially expressed proteins. For validation, 8 patients with MDD including 3 additional patients and 8 matched normal controls were analyzed. Results The quantitative proteomic studies identified 10 proteins that were consistently upregulated or downregulated in 5 MDD patients. ELISA yielded results consistent with the proteomic analysis for 3 proteins. Expression levels were significantly different between normal controls and MDD patients. The 3 proteins were ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4 and complement component 1qC, which were upregulated during the depressive status. The depressive status could be distinguished from the euthymic status from the ROC curves for these proteins, and this discrimination was enhanced when all 3 proteins were analyzed together. Conclusion This is the first proteomic study in MDD patients to compare intra-individual differences dependent on mood. This technique could be a useful approach to identify MDD biomarkers, but requires additional proteomic studies for validation. PMID:25866527
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Kirkwood, Kathryn J.; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I.
2013-01-01
Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community. PMID:24043423
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Kirkwood, Kathryn J; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I
2013-12-01
Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community.
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Placental Proteomics: A Shortcut to Biological Insight
Robinson, John M.; Vandré, Dale D.; Ackerman, William E.
2012-01-01
Proteomics analysis of biological samples has the potential to identify novel protein expression patterns and/or changes in protein expression patterns in different developmental or disease states. An important component of successful proteomics research, at least in its present form, is to reduce the complexity of the sample if it is derived from cells or tissues. One method to simplify complex tissues is to focus on a specific, highly purified sub-proteome. Using this approach we have developed methods to prepare highly enriched fractions of the apical plasma membrane of the syncytiotrophoblast. Through proteomics analysis of this fraction we have identified over five hundred proteins several of which were previously not known to reside in the syncytiotrophoblast. Herein, we focus on two of these, dysferlin and myoferlin. These proteins, largely known from studies of skeletal muscle, may not have been found in the human placenta were it not for discovery-based proteomics analysis. This new knowledge, acquired through a discovery-driven approach, can now be applied for the generation of hypothesis-based experimentation. Thus discovery-based and hypothesis-based research are complimentary approaches that when coupled together can hasten scientific discoveries. PMID:19070895
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Proteomics boosts translational and clinical microbiology.
Del Chierico, F; Petrucca, A; Vernocchi, P; Bracaglia, G; Fiscarelli, E; Bernaschi, P; Muraca, M; Urbani, A; Putignani, L
2014-01-31
The application of proteomics to translational and clinical microbiology is one of the most advanced frontiers in the management and control of infectious diseases and in the understanding of complex microbial systems within human fluids and districts. This new approach aims at providing, by dedicated bioinformatic pipelines, a thorough description of pathogen proteomes and their interactions within the context of human host ecosystems, revolutionizing the vision of infectious diseases in biomedicine and approaching new viewpoints in both diagnostic and clinical management of the patient. Indeed, in the last few years, many laboratories have matured a series of advanced proteomic applications, aiming at providing individual proteome charts of pathogens, with respect to their morph and/or cell life stages, antimicrobial or antimycotic resistance profiling, epidemiological dispersion. Herein, we aim at reviewing the current state-of-the-art on proteomic protocols designed and set-up for translational and diagnostic microbiological purposes, from axenic pathogens' characterization to microbiota ecosystems' full description. The final goal is to describe applications of the most common MALDI-TOF MS platforms to advanced diagnostic issues related to emerging infections, increasing of fastidious bacteria, and generation of patient-tailored phylotypes. This article is part of a Special Issue entitled: Trends in Microbial Proteomics. © 2013. Published by Elsevier B.V. All rights reserved.
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Percy, Andrew J; Yang, Juncong; Chambers, Andrew G; Mohammed, Yassene; Miliotis, Tasso; Borchers, Christoph H
2016-01-01
Quantitative mass spectrometry (MS)-based approaches are emerging as a core technology for addressing health-related queries in systems biology and in the biomedical and clinical fields. In several 'omics disciplines (proteomics included), an approach centered on selected or multiple reaction monitoring (SRM or MRM)-MS with stable isotope-labeled standards (SIS), at the protein or peptide level, has emerged as the most precise technique for quantifying and screening putative analytes in biological samples. To enable the widespread use of MRM-based protein quantitation for disease biomarker assessment studies and its ultimate acceptance for clinical analysis, the technique must be standardized to facilitate precise and accurate protein quantitation. To that end, we have developed a number of kits for assessing method/platform performance, as well as for screening proposed candidate protein biomarkers in various human biofluids. Collectively, these kits utilize a bottom-up LC-MS methodology with SIS peptides as internal standards and quantify proteins using regression analysis of standard curves. This chapter details the methodology used to quantify 192 plasma proteins of high-to-moderate abundance (covers a 6 order of magnitude range from 31 mg/mL for albumin to 18 ng/mL for peroxidredoxin-2), and a 21-protein subset thereof. We also describe the application of this method to patient samples for biomarker discovery and verification studies. Additionally, we introduce our recently developed Qualis-SIS software, which is used to expedite the analysis and assessment of protein quantitation data in control and patient samples.
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Vítámvás, Pavel; Urban, Milan O.; Škodáček, Zbynek; Kosová, Klára; Pitelková, Iva; Vítámvás, Jan; Renaut, Jenny; Prášil, Ilja T.
2015-01-01
Barley cultivar Amulet was used to study the quantitative proteome changes through different drought conditions utilizing two-dimensional difference gel electrophoresis (2D-DIGE). Plants were cultivated for 10 days under different drought conditions. To obtain control and differentially drought-treated plants, the soil water content was kept at 65, 35, and 30% of soil water capacity (SWC), respectively. Osmotic potential, water saturation deficit, 13C discrimination, and dehydrin accumulation were monitored during sampling of the crowns for proteome analysis. Analysis of the 2D-DIGE gels revealed 105 differentially abundant spots; most were differentially abundant between the controls and drought-treated plants, and 25 spots displayed changes between both drought conditions. Seventy-six protein spots were successfully identified by tandem mass spectrometry. The most frequent functional categories of the identified proteins can be put into the groups of: stress-associated proteins, amino acid metabolism, carbohydrate metabolism, as well as DNA and RNA regulation and processing. Their possible role in the response of barley to drought stress is discussed. Our study has shown that under drought conditions barley cv. Amulet decreased its growth and developmental rates, displayed a shift from aerobic to anaerobic metabolism, and exhibited increased levels of several protective proteins. Comparison of the two drought treatments revealed plant acclimation to milder drought (35% SWC); but plant damage under more severe drought treatment (30% SWC). The results obtained revealed that cv. Amulet is sensitive to drought stress. Additionally, four spots revealing a continuous and significant increase with decreasing SWC (UDP-glucose 6-dehydrogenase, glutathione peroxidase, and two non-identified) could be good candidates for testing of their protein phenotyping capacity together with proteins that were significantly distinguished in both drought treatments. PMID:26175745
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Gianazza, Erica; Tremoli, Elena; Banfi, Cristina
2014-12-01
Selected reaction monitoring, also known as multiple reaction monitoring, is a powerful targeted mass spectrometry approach for a confident quantitation of proteins/peptides in complex biological samples. In recent years, its optimization and application have become pivotal and of great interest in clinical research to derive useful outcomes for patient care. Thus, selected reaction monitoring/multiple reaction monitoring is now used as a highly sensitive and selective method for the evaluation of protein abundances and biomarker verification with potential applications in medical screening. This review describes technical aspects for the development of a robust multiplex assay and discussing its recent applications in cardiovascular proteomics: verification of promising disease candidates to select only the highest quality peptides/proteins for a preclinical validation, as well as quantitation of protein isoforms and post-translational modifications.
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Quantitative Profiling of Brain Lipid Raft Proteome in a Mouse Model of Fragile X Syndrome
Kalinowska, Magdalena; Castillo, Catherine; Francesconi, Anna
2015-01-01
Fragile X Syndrome, a leading cause of inherited intellectual disability and autism, arises from transcriptional silencing of the FMR1 gene encoding an RNA-binding protein, Fragile X Mental Retardation Protein (FMRP). FMRP can regulate the expression of approximately 4% of brain transcripts through its role in regulation of mRNA transport, stability and translation, thus providing a molecular rationale for its potential pleiotropic effects on neuronal and brain circuitry function. Several intracellular signaling pathways are dysregulated in the absence of FMRP suggesting that cellular deficits may be broad and could result in homeostatic changes. Lipid rafts are specialized regions of the plasma membrane, enriched in cholesterol and glycosphingolipids, involved in regulation of intracellular signaling. Among transcripts targeted by FMRP, a subset encodes proteins involved in lipid biosynthesis and homeostasis, dysregulation of which could affect the integrity and function of lipid rafts. Using a quantitative mass spectrometry-based approach we analyzed the lipid raft proteome of Fmr1 knockout mice, an animal model of Fragile X syndrome, and identified candidate proteins that are differentially represented in Fmr1 knockout mice lipid rafts. Furthermore, network analysis of these candidate proteins reveals connectivity between them and predicts functional connectivity with genes encoding components of myelin sheath, axonal processes and growth cones. Our findings provide insight to aid identification of molecular and cellular dysfunctions arising from Fmr1 silencing and for uncovering shared pathologies between Fragile X syndrome and other autism spectrum disorders. PMID:25849048
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Cologna, Nicholas de Mojana di; Gómez-Mendoza, Diana Paola; Zanoelo, Fabiana Fonseca; Giannesi, Giovana Cristina; Guimarães, Nelciele Cavalieri de Alencar; Moreira, Leonora Rios de Souza; Filho, Edivaldo Ximenes Ferreira; Ricart, Carlos André Ornelas
2018-02-01
Filamentous fungal secretomes comprise highly dynamic sets of proteins, including multiple carbohydrate active enzymes (CAZymes) which are able to hydrolyze plant biomass polysaccharides into products of biotechnological interest such as fermentable sugars. In recent years, proteomics has been used to identify and quantify enzymatic and non-enzymatic polypeptides present in secretomes of several fungi species. The resulting data have widened the scientific understanding of the way filamentous fungi perform biomass degradation and offered novel perspectives for biotechnological applications. The present review discusses proteomics approaches that have been applied to the study of fungal secretomes, focusing on two of the most studied filamentous fungi genera: Trichoderma and Aspergillus. Copyright © 2017 Elsevier Inc. All rights reserved.
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Pasquali, Matias; Serchi, Tommaso; Planchon, Sebastien; Renaut, Jenny
2017-01-01
The two-dimensional difference gel electrophoresis method is a valuable approach for proteomics. The method, using cyanine fluorescent dyes, allows the co-migration of multiple protein samples in the same gel and their simultaneous detection, thus reducing experimental and analytical time. 2D-DIGE, compared to traditional post-staining 2D-PAGE protocols (e.g., colloidal Coomassie or silver nitrate), provides faster and more reliable gel matching, limiting the impact of gel to gel variation, and allows also a good dynamic range for quantitative comparisons. By the use of internal standards, it is possible to normalize for experimental variations in spot intensities and gel patterns. Here we describe the experimental steps we follow in our routine 2D-DIGE procedure that we then apply to multiple biological questions.
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Deng, Ning; Li, Zhenye; Pan, Chao; Duan, Huilong
2015-01-01
Study of complex proteome brings forward higher request for the quantification method using mass spectrometry technology. In this paper, we present a mass spectrometry label-free quantification tool for complex proteomes, called freeQuant, which integrated quantification with functional analysis effectively. freeQuant consists of two well-integrated modules: label-free quantification and functional analysis with biomedical knowledge. freeQuant supports label-free quantitative analysis which makes full use of tandem mass spectrometry (MS/MS) spectral count, protein sequence length, shared peptides, and ion intensity. It adopts spectral count for quantitative analysis and builds a new method for shared peptides to accurately evaluate abundance of isoforms. For proteins with low abundance, MS/MS total ion count coupled with spectral count is included to ensure accurate protein quantification. Furthermore, freeQuant supports the large-scale functional annotations for complex proteomes. Mitochondrial proteomes from the mouse heart, the mouse liver, and the human heart were used to evaluate the usability and performance of freeQuant. The evaluation showed that the quantitative algorithms implemented in freeQuant can improve accuracy of quantification with better dynamic range.
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Zhang, Hengwei; Recker, Robert; Lee, Wai-Nang Paul; Xiao, Gary Guishan
2010-01-01
Osteoporosis is prevalent among the elderly and is a major cause of bone fracture in this population. Bone integrity is maintained by the dynamic processes of bone resorption and bone formation (bone remodeling). Osteoporosis results when there is an imbalance of the two counteracting processes. Bone mineral density, measured by dual-energy x-ray absorptiometry has been the primary method to assess fracture risk for decades. Recent studies demonstrated that measurement of bone turnover markers allows for a dynamic assessment of bone remodeling, while imaging techniques, such as dual-energy x-ray absorptiometry, do not. The application of proteomics has permitted discoveries of new, sensitive, bone turnover markers, which provide unique information for clinical diagnosis and treatment of patients with bone diseases. This review summarizes the recent findings of proteomic studies on bone diseases, properties of mesenchymal stem cells with high expansion rates and osteoblast and osteoclast differentiation, with emphasis on the role of quantitative proteomics in the study of signaling dynamics, biomarkers and discovery of therapeutic targets. PMID:20121480
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Proteomic Analysis of the Human Skin Proteome after In Vivo Treatment with Sodium Dodecyl Sulphate
Parkinson, Erika; Skipp, Paul; Aleksic, Maja; Garrow, Andrew; Dadd, Tony; Hughes, Michael; Clough, Geraldine; O′Connor, C. David
2014-01-01
Background Skin has a variety of functions that are incompletely understood at the molecular level. As the most accessible tissue in the body it often reveals the first signs of inflammation or infection and also represents a potentially valuable source of biomarkers for several diseases. In this study we surveyed the skin proteome qualitatively using gel electrophoresis, liquid chromatography tandem mass spectrometry (GeLC-MS/MS) and quantitatively using an isobaric tagging strategy (iTRAQ) to characterise the response of human skin following exposure to sodium dodecyl sulphate (SDS). Results A total of 653 skin proteins were assigned, 159 of which were identified using GeLC-MS/MS and 616 using iTRAQ, representing the most comprehensive proteomic study in human skin tissue. Statistical analysis of the available iTRAQ data did not reveal any significant differences in the measured skin proteome after 4 hours exposure to the model irritant SDS. Conclusions This study represents the first step in defining the critical response to an irritant at the level of the proteome and provides a valuable resource for further studies at the later stages of irritant exposure. PMID:24849295
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Hammad, Ghania; Legrain, Yona; Touat-Hamici, Zahia; Duhieu, Stéphane; Cornu, David; Bulteau, Anne-Laure; Chavatte, Laurent
2018-01-20
Selenoproteins are essential components of antioxidant defense, redox homeostasis, and cell signaling in mammals, where selenium is found in the form of a rare amino acid, selenocysteine. Selenium, which is often limited both in food intake and cell culture media, is a strong regulator of selenoprotein expression and selenoenzyme activity. Aging is a slow, complex, and multifactorial process, resulting in a gradual and irreversible decline of various functions of the body. Several cellular aspects of organismal aging are recapitulated in the replicative senescence of cultured human diploid fibroblasts, such as embryonic lung fibroblast WI-38 cells. We previously reported that the long-term growth of young WI-38 cells with high (supplemented), moderate (control), or low (depleted) concentrations of selenium in the culture medium impacts their replicative lifespan, due to rapid changes in replicative senescence-associated markers and signaling pathways. In order to gain insight into the molecular link between selenium levels and replicative senescence, in the present work, we have applied a quantitative proteomic approach based on 2-Dimensional Differential in-Gel Electrophoresis (2D-DIGE) to the study of young and presenescent cells grown in selenium-supplemented, control, or depleted media. Applying a restrictive cut-off (spot intensity ±50% and a p value < 0.05) to the 2D-DIGE analyses revealed 81 differentially expressed protein spots, from which 123 proteins of interest were identified by mass spectrometry. We compared the changes in protein abundance for three different conditions: (i) spots varying between young and presenescent cells, (ii) spots varying in response to selenium concentration in young cells, and (iii) spots varying in response to selenium concentration in presenescent cells. Interestingly, a 72% overlap between the impact of senescence and selenium was observed in our proteomic results, demonstrating a strong interplay between selenium
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Proteomic and transcriptomic analysis of lung tissue in OVA-challenged mice.
Lee, Yongjin; Hwang, Yun-Ho; Kim, Kwang-Jin; Park, Ae-Kyung; Paik, Man-Jeong; Kim, Seong Hwan; Lee, Su Ui; Yee, Sung-Tae; Son, Young-Jin
2018-01-01
Asthma is a long term inflammatory disease of the airway of lungs characterized by variable airflow obstruction and bronchospasm. Asthma is caused by a complex combination of environmental and genetic interactions. In this study, we conducted proteomic analysis of samples derived from control and OVA challenged mice for environmental respiratory disease by using 2-D gel electrophoresis. In addition, we explored the genes associated with the environmental substances that cause respiratory disease and conducted RNA-seq by next-generation sequencing. Proteomic analysis revealed 7 up-regulated (keratin KB40, CRP, HSP27, chaperonin containing TCP-1, TCP-10, keratin, and albumin) and 3 down-regulated proteins (PLC-α, PLA2, and precursor ApoA-1). The expression diversity of many genes was found in the lung tissue of OVA challenged moue by RNA-seq. 146 genes were identified as significantly differentially expressed by OVA treatment, and 118 genes of the 146 differentially expressed genes were up-regulated and 28 genes were downregulated. These genes were related to inflammation, mucin production, and airway remodeling. The results presented herein enable diagnosis and the identification of quantitative markers to monitor the progression of environmental respiratory disease using proteomics and genomic approaches.
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LFQuant: a label-free fast quantitative analysis tool for high-resolution LC-MS/MS proteomics data.
Zhang, Wei; Zhang, Jiyang; Xu, Changming; Li, Ning; Liu, Hui; Ma, Jie; Zhu, Yunping; Xie, Hongwei
2012-12-01
Database searching based methods for label-free quantification aim to reconstruct the peptide extracted ion chromatogram based on the identification information, which can limit the search space and thus make the data processing much faster. The random effect of the MS/MS sampling can be remedied by cross-assignment among different runs. Here, we present a new label-free fast quantitative analysis tool, LFQuant, for high-resolution LC-MS/MS proteomics data based on database searching. It is designed to accept raw data in two common formats (mzXML and Thermo RAW), and database search results from mainstream tools (MASCOT, SEQUEST, and X!Tandem), as input data. LFQuant can handle large-scale label-free data with fractionation such as SDS-PAGE and 2D LC. It is easy to use and provides handy user interfaces for data loading, parameter setting, quantitative analysis, and quantitative data visualization. LFQuant was compared with two common quantification software packages, MaxQuant and IDEAL-Q, on the replication data set and the UPS1 standard data set. The results show that LFQuant performs better than them in terms of both precision and accuracy, and consumes significantly less processing time. LFQuant is freely available under the GNU General Public License v3.0 at http://sourceforge.net/projects/lfquant/. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Jiang, Xiao-Sheng; Dai, Jie; Sheng, Quan-Hu; Zhang, Lei; Xia, Qi-Chang; Wu, Jia-Rui; Zeng, Rong
2005-01-01
Subcellular proteomics, as an important step to functional proteomics, has been a focus in proteomic research. However, the co-purification of "contaminating" proteins has been the major problem in all the subcellular proteomic research including all kinds of mitochondrial proteome research. It is often difficult to conclude whether these "contaminants" represent true endogenous partners or artificial associations induced by cell disruption or incomplete purification. To solve such a problem, we applied a high-throughput comparative proteome experimental strategy, ICAT approach performed with two-dimensional LC-MS/MS analysis, coupled with combinational usage of different bioinformatics tools, to study the proteome of rat liver mitochondria prepared with traditional centrifugation (CM) or further purified with a Nycodenz gradient (PM). A total of 169 proteins were identified and quantified convincingly in the ICAT analysis, in which 90 proteins have an ICAT ratio of PM:CM>1.0, while another 79 proteins have an ICAT ratio of PM:CM<1.0. Almost all the proteins annotated as mitochondrial according to Swiss-Prot annotation, bioinformatics prediction, and literature reports have a ratio of PM:CM>1.0, while proteins annotated as extracellular or secreted, cytoplasmic, endoplasmic reticulum, ribosomal, and so on have a ratio of PM:CM<1.0. Catalase and AP endonuclease 1, which have been known as peroxisomal and nuclear, respectively, have shown a ratio of PM:CM>1.0, confirming the reports about their mitochondrial location. Moreover, the 125 proteins with subcellular location annotation have been used as a testing dataset to evaluate the efficiency for ascertaining mitochondrial proteins by ICAT analysis and the bioinformatics tools such as PSORT, TargetP, SubLoc, MitoProt, and Predotar. The results indicated that ICAT analysis coupled with combinational usage of different bioinformatics tools could effectively ascertain mitochondrial proteins and distinguish contaminant
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Komatsu, Setsuko; Wang, Xin; Yin, Xiaojian; Nanjo, Yohei; Ohyanagi, Hajime; Sakata, Katsumi
2017-06-23
The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max 'Enrei'). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. The Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all predicted proteins from
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Quantitative Proteomic Profiling of Early and Late Responses to Salicylic Acid in Cucumber Leaves
Li, Liang; Shang, Qing-Mao
2016-01-01
Salicylic acid (SA) is an important phytohormone that plays vital regulatory roles in plant growth, development, and stress responses. However, studies on the molecular mechanism of SA, especially during the early SA responses, are lagging behind. In this study, we initiated a comprehensive isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis to explore the early and late SA-responsive proteins in leaves of cucumber (Cucumis sativus L.) seedlings. Upon SA application through the roots, endogenous SA accumulated in cucumber leaves. By assaying the changes in marker gene expression and photosynthetic rate, we collected samples at 12 h and 72 h post treatment (hpt) to profile the early and late SA responsiveness, respectively. The iTRAQ assay followed by tandem mass spectrometry revealed 135 differentially expressed proteins (DEPs) at 12 hpt and 301 DEPs at 72 hpt. The functional categories for these SA-responsive proteins included in a variety of biochemical processes, including photosynthesis, redox homeostasis, carbohydrate and energy metabolism, lipid metabolism, transport, protein folding and modification, proteolysis, cell wall organization, and the secondary phenylpropanoid pathway. Conclusively, based on the abundant changes of these DEPs, together with their putative functions, we proposed a possible SA-responsive protein network. It appears that SA could elicit reactive oxygen species (ROS) production via enhancing the photosynthetic electron transferring, and then confer some growth-promoting and stress-priming effects on cells during the late phase, including enhanced photosynthesis and ROS scavenging, altered carbon metabolic flux for the biosynthesis of amino acids and nucleotides, and cell wall reorganization. Overall, the present iTRAQ assay provides higher proteome coverage and deepened our understanding of the molecular basis of SA-responses. PMID:27551830
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Linking the proteins--elucidation of proteome-scale networks using mass spectrometry.
Pflieger, Delphine; Gonnet, Florence; de la Fuente van Bentem, Sergio; Hirt, Heribert; de la Fuente, Alberto
2011-01-01
Proteomes are intricate. Typically, thousands of proteins interact through physical association and post-translational modifications (PTMs) to give rise to the emergent functions of cells. Understanding these functions requires one to study proteomes as "systems" rather than collections of individual protein molecules. The abstraction of the interacting proteome to "protein networks" has recently gained much attention, as networks are effective representations, that lose specific molecular details, but provide the ability to see the proteome as a whole. Mostly two aspects of the proteome have been represented by network models: proteome-wide physical protein-protein-binding interactions organized into Protein Interaction Networks (PINs), and proteome-wide PTM relations organized into Protein Signaling Networks (PSNs). Mass spectrometry (MS) techniques have been shown to be essential to reveal both of these aspects on a proteome-wide scale. Techniques such as affinity purification followed by MS have been used to elucidate protein-protein interactions, and MS-based quantitative phosphoproteomics is critical to understand the structure and dynamics of signaling through the proteome. We here review the current state-of-the-art MS-based analytical pipelines for the purpose to characterize proteome-scale networks. Copyright © 2010 Wiley Periodicals, Inc.
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Navarrete-Perea, José; Isasa, Marta; Paulo, Joao A.; Corral-Corral, Ricardo; Flores-Bautista, Jeanette; Hernández-Téllez, Beatriz; Bobes, Raúl J.; Fragoso, Gladis; Sciutto, Edda; Soberón, Xavier; Gygi, Steven P.; Laclette, Juan P.
2017-01-01
In human and porcine cysticercosis caused by the tapeworm Taenia solium, the larval stage (cysts) can infest several tissues including the central nervous system (CNS) and the skeletal muscles (SM). The cyst’s proteomics changes associated with the tissue localization in the host tissues have been poorly studied. Quantitative multiplexed proteomics has the power to evaluate global proteome changes in response to different conditions. Here, using a TMT-multiplexed strategy we identified and quantified over 4,200 proteins in cysts obtained from the SM and CNS of pigs, of which 891 were host proteins. To our knowledge, this is the most extensive intermixing of host and parasite proteins reported for tapeworm infections.Several antigens in cysticercosis, i.e., GP50, paramyosin and a calcium-binding protein were enriched in skeletal muscle cysts. Our results suggested the occurrence of tissue-enriched antigen that could be useful in the improvement of the immunodiagnosis for cysticercosis. Using several algorithms for epitope detection, we selected 42 highly antigenic proteins enriched for each tissue localization of the cysts. Taking into account the fold changes and the antigen/epitope contents, we selected 10 proteins and produced synthetic peptides from the best epitopes. Nine peptides were recognized by serum antibodies of cysticercotic pigs, suggesting that those peptides are antigens. Mixtures of peptides derived from SM and CNS cysts yielded better results than mixtures of peptides derived from a single tissue location, however the identification of the ‘optimal’ tissue-enriched antigens remains to be discovered. Through machine learning technologies, we determined that a reliable immunodiagnostic test for porcine cysticercosis required at least five different antigenic determinants. PMID:28945737
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Navarrete-Perea, José; Isasa, Marta; Paulo, Joao A; Corral-Corral, Ricardo; Flores-Bautista, Jeanette; Hernández-Téllez, Beatriz; Bobes, Raúl J; Fragoso, Gladis; Sciutto, Edda; Soberón, Xavier; Gygi, Steven P; Laclette, Juan P
2017-09-01
In human and porcine cysticercosis caused by the tapeworm Taenia solium, the larval stage (cysts) can infest several tissues including the central nervous system (CNS) and the skeletal muscles (SM). The cyst's proteomics changes associated with the tissue localization in the host tissues have been poorly studied. Quantitative multiplexed proteomics has the power to evaluate global proteome changes in response to different conditions. Here, using a TMT-multiplexed strategy we identified and quantified over 4,200 proteins in cysts obtained from the SM and CNS of pigs, of which 891 were host proteins. To our knowledge, this is the most extensive intermixing of host and parasite proteins reported for tapeworm infections.Several antigens in cysticercosis, i.e., GP50, paramyosin and a calcium-binding protein were enriched in skeletal muscle cysts. Our results suggested the occurrence of tissue-enriched antigen that could be useful in the improvement of the immunodiagnosis for cysticercosis. Using several algorithms for epitope detection, we selected 42 highly antigenic proteins enriched for each tissue localization of the cysts. Taking into account the fold changes and the antigen/epitope contents, we selected 10 proteins and produced synthetic peptides from the best epitopes. Nine peptides were recognized by serum antibodies of cysticercotic pigs, suggesting that those peptides are antigens. Mixtures of peptides derived from SM and CNS cysts yielded better results than mixtures of peptides derived from a single tissue location, however the identification of the 'optimal' tissue-enriched antigens remains to be discovered. Through machine learning technologies, we determined that a reliable immunodiagnostic test for porcine cysticercosis required at least five different antigenic determinants.
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Magdalinou, N K; Noyce, A J; Pinto, R; Lindstrom, E; Holmén-Larsson, J; Holtta, M; Blennow, K; Morris, H R; Skillbäck, T; Warner, T T; Lees, A J; Pike, I; Ward, M; Zetterberg, H; Gobom, J
2017-04-01
Neurodegenerative parkinsonian syndromes have significant clinical and pathological overlap, making early diagnosis difficult. Cerebrospinal fluid (CSF) biomarkers may aid the differentiation of these disorders, but other than α-synuclein and neurofilament light chain protein, which have limited diagnostic power, specific protein biomarkers remain elusive. To study disease mechanisms and identify possible CSF diagnostic biomarkers through discovery proteomics, which discriminate parkinsonian syndromes from healthy controls. CSF was collected consecutively from 134 participants; Parkinson's disease (n = 26), atypical parkinsonian syndromes (n = 78, including progressive supranuclear palsy (n = 36), multiple system atrophy (n = 28), corticobasal syndrome (n = 14)), and elderly healthy controls (n = 30). Participants were divided into a discovery and a validation set for analysis. The samples were subjected to tryptic digestion, followed by liquid chromatography-mass spectrometry analysis for identification and relative quantification by isobaric labelling. Candidate protein biomarkers were identified based on the relative abundances of the identified tryptic peptides. Their predictive performance was evaluated by analysis of the validation set. 79 tryptic peptides, derived from 26 proteins were found to differ significantly between atypical parkinsonism patients and controls. They included acute phase/inflammatory markers and neuronal/synaptic markers, which were respectively increased or decreased in atypical parkinsonism, while their levels in PD subjects were intermediate between controls and atypical parkinsonism. Using an unbiased proteomic approach, proteins were identified that were able to differentiate atypical parkinsonian syndrome patients from healthy controls. Our study indicates that markers that may reflect neuronal function and/or plasticity, such as the amyloid precursor protein, and inflammatory markers may hold future promise as
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Mohideen, Firaz; Paulo, Joao A; Ordureau, Alban; Gygi, Steve P; Harper, J Wade
2017-07-01
TNFα is a potent inducer of inflammation due to its ability to promote gene expression, in part via the NFκB pathway. Moreover, in some contexts, TNFα promotes Caspase-dependent apoptosis or RIPK1/RIPK3/MLKL-dependent necrosis. Engagement of the TNF Receptor Signaling Complex (TNF-RSC), which contains multiple kinase activities, promotes phosphorylation of several downstream components, including TAK1, IKKα/IKKβ, IκBα, and NFκB. However, immediate downstream phosphorylation events occurring in response to TNFα signaling are poorly understood at a proteome-wide level. Here we use Tandem Mass Tagging-based proteomics to quantitatively characterize acute TNFα-mediated alterations in the proteome and phosphoproteome with or without inhibition of the cIAP-dependent survival arm of the pathway with a SMAC mimetic. We identify and quantify over 8,000 phosphorylated peptides, among which are numerous known sites in the TNF-RSC, NFκB, and MAP kinase signaling systems, as well as numerous previously unrecognized phosphorylation events. Functional analysis of S320 phosphorylation in RIPK1 demonstrates a role for this event in suppressing its kinase activity, association with CASPASE-8 and FADD proteins, and subsequent necrotic cell death during inflammatory TNFα stimulation. This study provides a resource for further elucidation of TNFα-dependent signaling pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Xing, Tong; Wang, Chong; Zhao, Xue; Dai, Chen; Zhou, Guanghong; Xu, Xinglian
2017-04-05
The current study was designed to investigate changes in the protein profiles of pale, soft, and exudative (PSE)-like muscles of broilers subjected to transportation under high-temperature conditions, using isobaric tags for relative and absolute analysis quantitation (iTRAQ). Arbor Acres chickens (n = 112) were randomly divided into two treatments: unstressed control (CON) and 0.5 h of transport (T). Birds were transported according to a designed protocol. Pectoralis major (PM) muscle samples in the T group were collected and classified as normal (T-NOR) or PSE-like (T-PSE). Plasma activities of stress indicators, muscle microstructure, and proteome were measured. Results indicated that broilers in the T-PSE group exhibited higher activities of plasma stress indicators. The microstructure of T-PSE group showed a looser network and larger intercellular spaces in comparison to the other groups. Proteomic analysis, based on iTRAQ, revealed 29 differentially expressed proteins in the T-NOR and T-PSE groups that were involved in protein turnover, signal transduction, stress and defense, calcium handling, cell structure, and metabolism. In particular, proteins relating to the glycolysis pathway, calcium signaling, and molecular chaperones exhibited significant differences that may contribute to the inferior post-mortem meat quality. Overall, the proteomic results provide a further understanding of the mechanism of meat quality changes in response to stress.
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Muñoz-Gómez, Amalia; Corredor, Mauricio; Benítez-Páez, Alfonso; Peláez, Carlos
2014-01-01
Galleria mellonella has emerged as a potential invertebrate model for scrutinizing innate immunity. Larvae are easy to handle in host-pathogen assays. We undertook proteomics research in order to understand immune response in a heterologous host when challenged with microconidia of Fusarium oxysporum. The aim of this study was to investigate hemolymph proteins that were differentially expressed between control and immunized larvae sets, tested with F. oxysporum at two temperatures. The iTRAQ approach allowed us to observe the effects of immune challenges in a lucid and robust manner, identifying more than 50 proteins, 17 of them probably involved in the immune response. Changes in protein expression were statistically significant, especially when temperature was increased because this was notoriously affected by F. oxysporum 104 or 106 microconidia/mL. Some proteins were up-regulated upon immune fungal microconidia challenge when temperature changed from 25 to 37°C. After analysis of identified proteins by bioinformatics and meta-analysis, results revealed that they were involved in transport, immune response, storage, oxide-reduction and catabolism: 20 from G. mellonella, 20 from the Lepidoptera species and 19 spread across bacteria, protista, fungi and animal species. Among these, 13 proteins and 2 peptides were examined for their immune expression, and the hypothetical 3D structures of 2 well-known proteins, unannotated for G. mellonella, i.e., actin and CREBP, were resolved using peptides matched with Bombyx mori and Danaus plexippus, respectively. The main conclusion in this study was that iTRAQ tool constitutes a consistent method to detect proteins associated with the innate immune system of G. mellonella in response to infection caused by F. oxysporum. In addition, iTRAQ was a reliable quantitative proteomic approach to detect and quantify the expression levels of immune system proteins and peptides, in particular, it was found that 104 microconidia/mL at
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Singec, Ilyas; Crain, Andrew M; Hou, Junjie; Tobe, Brian T D; Talantova, Maria; Winquist, Alicia A; Doctor, Kutbuddin S; Choy, Jennifer; Huang, Xiayu; La Monaca, Esther; Horn, David M; Wolf, Dieter A; Lipton, Stuart A; Gutierrez, Gustavo J; Brill, Laurence M; Snyder, Evan Y
2016-09-13
Controlled differentiation of human embryonic stem cells (hESCs) can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs). This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites) provides unique molecular insights into pluripotency and neural lineage entry. Systems-level comparative analysis of proteins (e.g., transcription factors, epigenetic regulators, kinase families), phosphorylation sites, and numerous biological pathways allowed the identification of distinct signatures in pluripotent and multipotent cells. Furthermore, as predicted by the dataset, we functionally validated an autocrine/paracrine mechanism by demonstrating that the secreted protein midkine is a regulator of neural specification. This resource is freely available to the scientific community, including a searchable website, PluriProt. Published by Elsevier Inc.