HiQuant: Rapid Postquantification Analysis of Large-Scale MS-Generated Proteomics Data.

PubMed

Bryan, Kenneth; Jarboui, Mohamed-Ali; Raso, Cinzia; Bernal-Llinares, Manuel; McCann, Brendan; Rauch, Jens; Boldt, Karsten; Lynn, David J

2016-06-03

Recent advances in mass-spectrometry-based proteomics are now facilitating ambitious large-scale investigations of the spatial and temporal dynamics of the proteome; however, the increasing size and complexity of these data sets is overwhelming current downstream computational methods, specifically those that support the postquantification analysis pipeline. Here we present HiQuant, a novel application that enables the design and execution of a postquantification workflow, including common data-processing steps, such as assay normalization and grouping, and experimental replicate quality control and statistical analysis. HiQuant also enables the interpretation of results generated from large-scale data sets by supporting interactive heatmap analysis and also the direct export to Cytoscape and Gephi, two leading network analysis platforms. HiQuant may be run via a user-friendly graphical interface and also supports complete one-touch automation via a command-line mode. We evaluate HiQuant's performance by analyzing a large-scale, complex interactome mapping data set and demonstrate a 200-fold improvement in the execution time over current methods. We also demonstrate HiQuant's general utility by analyzing proteome-wide quantification data generated from both a large-scale public tyrosine kinase siRNA knock-down study and an in-house investigation into the temporal dynamics of the KSR1 and KSR2 interactomes. Download HiQuant, sample data sets, and supporting documentation at http://hiquant.primesdb.eu .

Tissue proteomics of the low-molecular weight proteome using an integrated cLC-ESI-QTOFMS approach.

PubMed

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.

Dual-color Proteomic Profiling of Complex Samples with a Microarray of 810 Cancer-related Antibodies*

PubMed Central

Schröder, Christoph; Jacob, Anette; Tonack, Sarah; Radon, Tomasz P.; Sill, Martin; Zucknick, Manuela; Rüffer, Sven; Costello, Eithne; Neoptolemos, John P.; Crnogorac-Jurcevic, Tatjana; Bauer, Andrea; Fellenberg, Kurt; Hoheisel, Jörg D.

2010-01-01

Antibody microarrays have the potential to enable comprehensive proteomic analysis of small amounts of sample material. Here, protocols are presented for the production, quality assessment, and reproducible application of antibody microarrays in a two-color mode with an array of 1,800 features, representing 810 antibodies that were directed at 741 cancer-related proteins. In addition to measures of array quality, we implemented indicators for the accuracy and significance of dual-color detection. Dual-color measurements outperform a single-color approach concerning assay reproducibility and discriminative power. In the analysis of serum samples, depletion of high-abundance proteins did not improve technical assay quality. On the contrary, depletion introduced a strong bias in protein representation. In an initial study, we demonstrated the applicability of the protocols to proteins derived from urine samples. We identified differences between urine samples from pancreatic cancer patients and healthy subjects and between sexes. This study demonstrates that biomedically relevant data can be produced. As demonstrated by the thorough quality analysis, the dual-color antibody array approach proved to be competitive with other proteomic techniques and comparable in performance to transcriptional microarray analyses. PMID:20164060

Refining comparative proteomics by spectral counting to account for shared peptides and multiple search engines

PubMed Central

Chen, Yao-Yi; Dasari, Surendra; Ma, Ze-Qiang; Vega-Montoto, Lorenzo J.; Li, Ming

2013-01-01

Spectral counting has become a widely used approach for measuring and comparing protein abundance in label-free shotgun proteomics. However, when analyzing complex samples, the ambiguity of matching between peptides and proteins greatly affects the assessment of peptide and protein inventories, differentiation, and quantification. Meanwhile, the configuration of database searching algorithms that assign peptides to MS/MS spectra may produce different results in comparative proteomic analysis. Here, we present three strategies to improve comparative proteomics through spectral counting. We show that comparing spectral counts for peptide groups rather than for protein groups forestalls problems introduced by shared peptides. We demonstrate the advantage and flexibility of this new method in two datasets. We present four models to combine four popular search engines that lead to significant gains in spectral counting differentiation. Among these models, we demonstrate a powerful vote counting model that scales well for multiple search engines. We also show that semi-tryptic searching outperforms tryptic searching for comparative proteomics. Overall, these techniques considerably improve protein differentiation on the basis of spectral count tables. PMID:22552787

Refining comparative proteomics by spectral counting to account for shared peptides and multiple search engines.

PubMed

Chen, Yao-Yi; Dasari, Surendra; Ma, Ze-Qiang; Vega-Montoto, Lorenzo J; Li, Ming; Tabb, David L

2012-09-01

Spectral counting has become a widely used approach for measuring and comparing protein abundance in label-free shotgun proteomics. However, when analyzing complex samples, the ambiguity of matching between peptides and proteins greatly affects the assessment of peptide and protein inventories, differentiation, and quantification. Meanwhile, the configuration of database searching algorithms that assign peptides to MS/MS spectra may produce different results in comparative proteomic analysis. Here, we present three strategies to improve comparative proteomics through spectral counting. We show that comparing spectral counts for peptide groups rather than for protein groups forestalls problems introduced by shared peptides. We demonstrate the advantage and flexibility of this new method in two datasets. We present four models to combine four popular search engines that lead to significant gains in spectral counting differentiation. Among these models, we demonstrate a powerful vote counting model that scales well for multiple search engines. We also show that semi-tryptic searching outperforms tryptic searching for comparative proteomics. Overall, these techniques considerably improve protein differentiation on the basis of spectral count tables.

Inter-lab proteomics: data mining in collaborative projects on the basis of the HUPO brain proteome project's pilot studies.

PubMed

Hamacher, Michael; Gröttrup, Bernd; Eisenacher, Martin; Marcus, Katrin; Park, Young Mok; Meyer, Helmut E; Kwon, Kyung-Hoon; Stephan, Christian

2011-01-01

Several projects were initiated by the Human Proteome Organisation (HUPO) focusing on the proteome analysis of distinct human organs. The initiative dedicated to the brain, its development and correlated diseases is the HUPO Brain Proteome Project (HUPO BPP). An objective data submission, storage, and reprocessing strategy have been established with the help of the results gained in a pilot study phase and within subsequent studies. The bioinformatic relevance of the data is drawn from the inter-laboratory comparisons as well as from the recalculation of all data sets submitted by the different groups. In the following, results of the single groups as well as the centralised reprocessing effort are summarised, demonstrating the added-value of this concerted work.

Studies of a biochemical factory: tomato trichome deep expressed sequence tag sequencing and proteomics.

PubMed

Schilmiller, Anthony L; Miner, Dennis P; Larson, Matthew; McDowell, Eric; Gang, David R; Wilkerson, Curtis; Last, Robert L

2010-07-01

Shotgun proteomics analysis allows hundreds of proteins to be identified and quantified from a single sample at relatively low cost. Extensive DNA sequence information is a prerequisite for shotgun proteomics, and it is ideal to have sequence for the organism being studied rather than from related species or accessions. While this requirement has limited the set of organisms that are candidates for this approach, next generation sequencing technologies make it feasible to obtain deep DNA sequence coverage from any organism. As part of our studies of specialized (secondary) metabolism in tomato (Solanum lycopersicum) trichomes, 454 sequencing of cDNA was combined with shotgun proteomics analyses to obtain in-depth profiles of genes and proteins expressed in leaf and stem glandular trichomes of 3-week-old plants. The expressed sequence tag and proteomics data sets combined with metabolite analysis led to the discovery and characterization of a sesquiterpene synthase that produces beta-caryophyllene and alpha-humulene from E,E-farnesyl diphosphate in trichomes of leaf but not of stem. This analysis demonstrates the utility of combining high-throughput cDNA sequencing with proteomics experiments in a target tissue. These data can be used for dissection of other biochemical processes in these specialized epidermal cells.

Studies of a Biochemical Factory: Tomato Trichome Deep Expressed Sequence Tag Sequencing and Proteomics1[W][OA

PubMed Central

Schilmiller, Anthony L.; Miner, Dennis P.; Larson, Matthew; McDowell, Eric; Gang, David R.; Wilkerson, Curtis; Last, Robert L.

2010-01-01

Shotgun proteomics analysis allows hundreds of proteins to be identified and quantified from a single sample at relatively low cost. Extensive DNA sequence information is a prerequisite for shotgun proteomics, and it is ideal to have sequence for the organism being studied rather than from related species or accessions. While this requirement has limited the set of organisms that are candidates for this approach, next generation sequencing technologies make it feasible to obtain deep DNA sequence coverage from any organism. As part of our studies of specialized (secondary) metabolism in tomato (Solanum lycopersicum) trichomes, 454 sequencing of cDNA was combined with shotgun proteomics analyses to obtain in-depth profiles of genes and proteins expressed in leaf and stem glandular trichomes of 3-week-old plants. The expressed sequence tag and proteomics data sets combined with metabolite analysis led to the discovery and characterization of a sesquiterpene synthase that produces β-caryophyllene and α-humulene from E,E-farnesyl diphosphate in trichomes of leaf but not of stem. This analysis demonstrates the utility of combining high-throughput cDNA sequencing with proteomics experiments in a target tissue. These data can be used for dissection of other biochemical processes in these specialized epidermal cells. PMID:20431087

Prestroke Proteomic Changes in Cerebral Microvessels in Stroke-Prone, Transgenic[hCETP]-Hyperlipidemic, Dahl Salt-Sensitive Hypertensive Rats

PubMed Central

Bergerat, Agnes; Decano, Julius; Wu, Chang-Jiun; Choi, Hyungwon; Nesvizhskii, Alexey I; Moran, Ann Marie; Ruiz-Opazo, Nelson; Steffen, Martin; Herrera, Victoria LM

2011-01-01

Stroke is the third leading cause of death in the United States with high rates of morbidity among survivors. The search to fill the unequivocal need for new therapeutic approaches would benefit from unbiased proteomic analyses of animal models of spontaneous stroke in the prestroke stage. Since brain microvessels play key roles in neurovascular coupling, we investigated prestroke microvascular proteome changes. Proteomic analysis of cerebral cortical microvessels (cMVs) was done by tandem mass spectrometry comparing two prestroke time points. Metaprotein-pathway analyses of proteomic spectral count data were done to identify risk factor–induced changes, followed by QSPEC-analyses of individual protein changes associated with increased stroke susceptibility. We report 26 cMV proteome profiles from male and female stroke-prone and non–stroke-prone rats at 2 months and 4.5 months of age prior to overt stroke events. We identified 1,934 proteins by two or more peptides. Metaprotein pathway analysis detected age-associated changes in energy metabolism and cell-to-microenvironment interactions, as well as sex-specific changes in energy metabolism and endothelial leukocyte transmigration pathways. Stroke susceptibility was associated independently with multiple protein changes associated with ischemia, angiogenesis or involved in blood brain barrier (BBB) integrity. Immunohistochemical analysis confirmed aquaporin-4 and laminin-α1 induction in cMVs, representative of proteomic changes with >65 Bayes factor (BF), associated with stroke susceptibility. Altogether, proteomic analysis demonstrates significant molecular changes in ischemic cerebral microvasculature in the prestroke stage, which could contribute to the observed model phenotype of microhemorrhages and postischemic hemorrhagic transformation. These pathways comprise putative targets for translational research of much needed novel diagnostic and therapeutic approaches for stroke. PMID:21519634

Comparative Proteome Analysis between High Lipid-Producing Strain Mucor circinelloides WJ11 and Low Lipid-Producing Strain CBS 277.49.

PubMed

Tang, Xin; Chen, Haiqin; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Song, Yuanda; Chen, Wei

2017-06-21

Mucor circinelloides is one of few oleaginous fungi that produces a useful oil rich in γ-linolenic acid, but it usually only produces <25% total lipid. Nevertheless, we isolated a new strain WJ11 that can produce up to 36% lipid of cell dry weight. In this study, we have systematically analyzed the global changes in protein levels between the high lipid-producing strain WJ11 and the low lipid-producing strain CBS 277.49 (15%, lipid/cell dry weight) at lipid accumulation phase through comparative proteome analysis. Proteome analysis demonstrated that the branched-chain amino acid and lysine metabolism, glycolytic pathway, and pentose phosphate pathway in WJ11 were up-regulated, while the activities of tricarboxylic acid cycle and branch point enzyme for synthesis of isoprenoids were retarded compared with CBS 277.49. The coordinated regulation at proteome level indicate that more acetyl-CoA and NADPH are provided for fatty acid biosynthesis in WJ11 compared with CBS 277.49.

Combined metabolome and proteome analysis of the mantle tissue from Pacific oyster Crassostrea gigas exposed to elevated pCO2.

PubMed

Wei, Lei; Wang, Qing; Ning, Xuanxuan; Mu, Changkao; Wang, Chunlin; Cao, Ruiwen; Wu, Huifeng; Cong, Ming; Li, Fei; Ji, Chenglong; Zhao, Jianmin

2015-03-01

Ocean acidification (OA) has been found to affect an array of normal physiological processes in mollusks, especially posing a significant threat to the fabrication process of mollusk shell. In the current study, the impact of exposure to elevated pCO2 condition was investigated in mantle tissue of Crassostrea gigas by an integrated metabolomic and proteomic approach. Analysis of metabolome and proteome revealed that elevated pCO2 could affect energy metabolism in oyster C. gigas, marked by differentially altered ATP, succinate, MDH, PEPCK and ALDH levels. Moreover, the up-regulated calponin-2, tropomyosins and myosin light chains indicated that elevated pCO2 probably caused disturbances in cytoskeleton structure in mantle tissue of oyster C. gigas. This work demonstrated that a combination of proteomics and metabolomics could provide important insights into the effects of OA at molecular levels. Copyright © 2014 Elsevier Inc. All rights reserved.

Spatially-Resolved Proteomics: Rapid Quantitative Analysis of Laser Capture Microdissected Alveolar Tissue Samples

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 clinical and biological applications.« less

Bacterial membrane proteomics.

PubMed

Poetsch, Ansgar; Wolters, Dirk

2008-10-01

About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.

Proteome-wide analysis and diel proteomic profiling of the cyanobacterium Arthrospira platensis PCC 8005.

PubMed

Matallana-Surget, Sabine; Derock, Jérémy; Leroy, Baptiste; Badri, Hanène; Deschoenmaeker, Frédéric; Wattiez, Ruddy

2014-01-01

The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation.

Proteome-Wide Analysis and Diel Proteomic Profiling of the Cyanobacterium Arthrospira platensis PCC 8005

PubMed Central

Matallana-Surget, Sabine; Derock, Jérémy; Leroy, Baptiste; Badri, Hanène; Deschoenmaeker, Frédéric; Wattiez, Ruddy

2014-01-01

The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation. PMID:24914774

QC-ART: A tool for real-time quality control assessment of mass spectrometry-based proteomics data.

PubMed

Stanfill, Bryan A; Nakayasu, Ernesto S; Bramer, Lisa M; Thompson, Allison M; Ansong, Charles K; Clauss, Therese; Gritsenko, Marina A; Monroe, Matthew E; Moore, Ronald J; Orton, Daniel J; Piehowski, Paul D; Schepmoes, Athena A; Smith, Richard D; Webb-Robertson, Bobbie-Jo; Metz, Thomas O

2018-04-17

Liquid chromatography-mass spectrometry (LC-MS)-based proteomics studies of large sample cohorts can easily require from months to years to complete. Acquiring consistent, high-quality data in such large-scale studies is challenging because of normal variations in instrumentation performance over time, as well as artifacts introduced by the samples themselves, such as those due to collection, storage and processing. Existing quality control methods for proteomics data primarily focus on post-hoc analysis to remove low-quality data that would degrade downstream statistics; they are not designed to evaluate the data in near real-time, which would allow for interventions as soon as deviations in data quality are detected.  In addition to flagging analyses that demonstrate outlier behavior, evaluating how the data structure changes over time can aide in understanding typical instrument performance or identify issues such as a degradation in data quality due to the need for instrument cleaning and/or re-calibration.  To address this gap for proteomics, we developed Quality Control Analysis in Real-Time (QC-ART), a tool for evaluating data as they are acquired in order to dynamically flag potential issues with instrument performance or sample quality.  QC-ART has similar accuracy as standard post-hoc analysis methods with the additional benefit of real-time analysis.  We demonstrate the utility and performance of QC-ART in identifying deviations in data quality due to both instrument and sample issues in near real-time for LC-MS-based plasma proteomics analyses of a sample subset of The Environmental Determinants of Diabetes in the Young cohort. We also present a case where QC-ART facilitated the identification of oxidative modifications, which are often underappreciated in proteomic experiments. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Automated image alignment for 2D gel electrophoresis in a high-throughput proteomics pipeline.

PubMed

Dowsey, Andrew W; Dunn, Michael J; Yang, Guang-Zhong

2008-04-01

The quest for high-throughput proteomics has revealed a number of challenges in recent years. Whilst substantial improvements in automated protein separation with liquid chromatography and mass spectrometry (LC/MS), aka 'shotgun' proteomics, have been achieved, large-scale open initiatives such as the Human Proteome Organization (HUPO) Brain Proteome Project have shown that maximal proteome coverage is only possible when LC/MS is complemented by 2D gel electrophoresis (2-DE) studies. Moreover, both separation methods require automated alignment and differential analysis to relieve the bioinformatics bottleneck and so make high-throughput protein biomarker discovery a reality. The purpose of this article is to describe a fully automatic image alignment framework for the integration of 2-DE into a high-throughput differential expression proteomics pipeline. The proposed method is based on robust automated image normalization (RAIN) to circumvent the drawbacks of traditional approaches. These use symbolic representation at the very early stages of the analysis, which introduces persistent errors due to inaccuracies in modelling and alignment. In RAIN, a third-order volume-invariant B-spline model is incorporated into a multi-resolution schema to correct for geometric and expression inhomogeneity at multiple scales. The normalized images can then be compared directly in the image domain for quantitative differential analysis. Through evaluation against an existing state-of-the-art method on real and synthetically warped 2D gels, the proposed analysis framework demonstrates substantial improvements in matching accuracy and differential sensitivity. High-throughput analysis is established through an accelerated GPGPU (general purpose computation on graphics cards) implementation. Supplementary material, software and images used in the validation are available at http://www.proteomegrid.org/rain/.

Processing Shotgun Proteomics Data on the Amazon Cloud with the Trans-Proteomic Pipeline*

PubMed Central

Slagel, Joseph; Mendoza, Luis; Shteynberg, David; Deutsch, Eric W.; Moritz, Robert L.

2015-01-01

Cloud computing, where scalable, on-demand compute cycles and storage are available as a service, has the potential to accelerate mass spectrometry-based proteomics research by providing simple, expandable, and affordable large-scale computing to all laboratories regardless of location or information technology expertise. We present new cloud computing functionality for the Trans-Proteomic Pipeline, a free and open-source suite of tools for the processing and analysis of tandem mass spectrometry datasets. Enabled with Amazon Web Services cloud computing, the Trans-Proteomic Pipeline now accesses large scale computing resources, limited only by the available Amazon Web Services infrastructure, for all users. The Trans-Proteomic Pipeline runs in an environment fully hosted on Amazon Web Services, where all software and data reside on cloud resources to tackle large search studies. In addition, it can also be run on a local computer with computationally intensive tasks launched onto the Amazon Elastic Compute Cloud service to greatly decrease analysis times. We describe the new Trans-Proteomic Pipeline cloud service components, compare the relative performance and costs of various Elastic Compute Cloud service instance types, and present on-line tutorials that enable users to learn how to deploy cloud computing technology rapidly with the Trans-Proteomic Pipeline. We provide tools for estimating the necessary computing resources and costs given the scale of a job and demonstrate the use of cloud enabled Trans-Proteomic Pipeline by performing over 1100 tandem mass spectrometry files through four proteomic search engines in 9 h and at a very low cost. PMID:25418363

Processing shotgun proteomics data on the Amazon cloud with the trans-proteomic pipeline.

PubMed

Slagel, Joseph; Mendoza, Luis; Shteynberg, David; Deutsch, Eric W; Moritz, Robert L

2015-02-01

Cloud computing, where scalable, on-demand compute cycles and storage are available as a service, has the potential to accelerate mass spectrometry-based proteomics research by providing simple, expandable, and affordable large-scale computing to all laboratories regardless of location or information technology expertise. We present new cloud computing functionality for the Trans-Proteomic Pipeline, a free and open-source suite of tools for the processing and analysis of tandem mass spectrometry datasets. Enabled with Amazon Web Services cloud computing, the Trans-Proteomic Pipeline now accesses large scale computing resources, limited only by the available Amazon Web Services infrastructure, for all users. The Trans-Proteomic Pipeline runs in an environment fully hosted on Amazon Web Services, where all software and data reside on cloud resources to tackle large search studies. In addition, it can also be run on a local computer with computationally intensive tasks launched onto the Amazon Elastic Compute Cloud service to greatly decrease analysis times. We describe the new Trans-Proteomic Pipeline cloud service components, compare the relative performance and costs of various Elastic Compute Cloud service instance types, and present on-line tutorials that enable users to learn how to deploy cloud computing technology rapidly with the Trans-Proteomic Pipeline. We provide tools for estimating the necessary computing resources and costs given the scale of a job and demonstrate the use of cloud enabled Trans-Proteomic Pipeline by performing over 1100 tandem mass spectrometry files through four proteomic search engines in 9 h and at a very low cost. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Parsing and Quantification of Raw Orbitrap Mass Spectrometer Data Using RawQuant.

PubMed

Kovalchik, Kevin A; Moggridge, Sophie; Chen, David D Y; Morin, Gregg B; Hughes, Christopher S

2018-06-01

Effective analysis of protein samples by mass spectrometry (MS) requires careful selection and optimization of a range of experimental parameters. As the output from the primary detection device, the "raw" MS data file can be used to gauge the success of a given sample analysis. However, the closed-source nature of the standard raw MS file can complicate effective parsing of the data contained within. To ease and increase the range of analyses possible, the RawQuant tool was developed to enable parsing of raw MS files derived from Thermo Orbitrap instruments to yield meta and scan data in an openly readable text format. RawQuant can be commanded to export user-friendly files containing MS 1 , MS 2 , and MS 3 metadata as well as matrices of quantification values based on isobaric tagging approaches. In this study, the utility of RawQuant is demonstrated in several scenarios: (1) reanalysis of shotgun proteomics data for the identification of the human proteome, (2) reanalysis of experiments utilizing isobaric tagging for whole-proteome quantification, and (3) analysis of a novel bacterial proteome and synthetic peptide mixture for assessing quantification accuracy when using isobaric tags. Together, these analyses successfully demonstrate RawQuant for the efficient parsing and quantification of data from raw Thermo Orbitrap MS files acquired in a range of common proteomics experiments. In addition, the individual analyses using RawQuant highlights parametric considerations in the different experimental sets and suggests targetable areas to improve depth of coverage in identification-focused studies and quantification accuracy when using isobaric tags.

An integrated native mass spectrometry and top-down proteomics method that connects sequence to structure and function of macromolecular complexes

NASA Astrophysics Data System (ADS)

Li, Huilin; Nguyen, Hong Hanh; Ogorzalek Loo, Rachel R.; Campuzano, Iain D. G.; Loo, Joseph A.

2018-02-01

Mass spectrometry (MS) has become a crucial technique for the analysis of protein complexes. Native MS has traditionally examined protein subunit arrangements, while proteomics MS has focused on sequence identification. These two techniques are usually performed separately without taking advantage of the synergies between them. Here we describe the development of an integrated native MS and top-down proteomics method using Fourier-transform ion cyclotron resonance (FTICR) to analyse macromolecular protein complexes in a single experiment. We address previous concerns of employing FTICR MS to measure large macromolecular complexes by demonstrating the detection of complexes up to 1.8 MDa, and we demonstrate the efficacy of this technique for direct acquirement of sequence to higher-order structural information with several large complexes. We then summarize the unique functionalities of different activation/dissociation techniques. The platform expands the ability of MS to integrate proteomics and structural biology to provide insights into protein structure, function and regulation.

Elucidation of cross-species proteomic effects in human and hominin bone proteome identification through a bioinformatics experiment.

PubMed

Welker, F

2018-02-20

The study of ancient protein sequences is increasingly focused on the analysis of older samples, including those of ancient hominins. The analysis of such ancient proteomes thereby potentially suffers from "cross-species proteomic effects": the loss of peptide and protein identifications at increased evolutionary distances due to a larger number of protein sequence differences between the database sequence and the analyzed organism. Error-tolerant proteomic search algorithms should theoretically overcome this problem at both the peptide and protein level; however, this has not been demonstrated. If error-tolerant searches do not overcome the cross-species proteomic issue then there might be inherent biases in the identified proteomes. Here, a bioinformatics experiment is performed to test this using a set of modern human bone proteomes and three independent searches against sequence databases at increasing evolutionary distances: the human (0 Ma), chimpanzee (6-8 Ma) and orangutan (16-17 Ma) reference proteomes, respectively. Incorrectly suggested amino acid substitutions are absent when employing adequate filtering criteria for mutable Peptide Spectrum Matches (PSMs), but roughly half of the mutable PSMs were not recovered. As a result, peptide and protein identification rates are higher in error-tolerant mode compared to non-error-tolerant searches but did not recover protein identifications completely. Data indicates that peptide length and the number of mutations between the target and database sequences are the main factors influencing mutable PSM identification. The error-tolerant results suggest that the cross-species proteomics problem is not overcome at increasing evolutionary distances, even at the protein level. Peptide and protein loss has the potential to significantly impact divergence dating and proteome comparisons when using ancient samples as there is a bias towards the identification of conserved sequences and proteins. Effects are minimized between moderately divergent proteomes, as indicated by almost complete recovery of informative positions in the search against the chimpanzee proteome (≈90%, 6-8 Ma). This provides a bioinformatic background to future phylogenetic and proteomic analysis of ancient hominin proteomes, including the future description of novel hominin amino acid sequences, but also has negative implications for the study of fast-evolving proteins in hominins, non-hominin animals, and ancient bacterial proteins in evolutionary contexts.

Advances in mass spectrometry-based cancer research and analysis: from cancer proteomics to clinical diagnostics.

PubMed

Timms, John F; Hale, Oliver J; Cramer, Rainer

2016-06-01

The last 20 years have seen significant improvements in the analytical capabilities of biological mass spectrometry (MS). Studies using advanced MS have resulted in new insights into cell biology and the etiology of diseases as well as its use in clinical applications. This review discusses recent developments in MS-based technologies and their cancer-related applications with a focus on proteomics. It also discusses the issues around translating the research findings to the clinic and provides an outline of where the field is moving. Expert commentary: Proteomics has been problematic to adapt for the clinical setting. However, MS-based techniques continue to demonstrate potential in novel clinical uses beyond classical cancer proteomics.

Interlaboratory Study Characterizing a Yeast Performance Standard for Benchmarking LC-MS Platform Performance*

PubMed Central

Paulovich, Amanda G.; Billheimer, Dean; Ham, Amy-Joan L.; Vega-Montoto, Lorenzo; Rudnick, Paul A.; Tabb, David L.; Wang, Pei; Blackman, Ronald K.; Bunk, David M.; Cardasis, Helene L.; Clauser, Karl R.; Kinsinger, Christopher R.; Schilling, Birgit; Tegeler, Tony J.; Variyath, Asokan Mulayath; Wang, Mu; Whiteaker, Jeffrey R.; Zimmerman, Lisa J.; Fenyo, David; Carr, Steven A.; Fisher, Susan J.; Gibson, Bradford W.; Mesri, Mehdi; Neubert, Thomas A.; Regnier, Fred E.; Rodriguez, Henry; Spiegelman, Cliff; Stein, Stephen E.; Tempst, Paul; Liebler, Daniel C.

2010-01-01

Optimal performance of LC-MS/MS platforms is critical to generating high quality proteomics data. Although individual laboratories have developed quality control samples, there is no widely available performance standard of biological complexity (and associated reference data sets) for benchmarking of platform performance for analysis of complex biological proteomes across different laboratories in the community. Individual preparations of the yeast Saccharomyces cerevisiae proteome have been used extensively by laboratories in the proteomics community to characterize LC-MS platform performance. The yeast proteome is uniquely attractive as a performance standard because it is the most extensively characterized complex biological proteome and the only one associated with several large scale studies estimating the abundance of all detectable proteins. In this study, we describe a standard operating protocol for large scale production of the yeast performance standard and offer aliquots to the community through the National Institute of Standards and Technology where the yeast proteome is under development as a certified reference material to meet the long term needs of the community. Using a series of metrics that characterize LC-MS performance, we provide a reference data set demonstrating typical performance of commonly used ion trap instrument platforms in expert laboratories; the results provide a basis for laboratories to benchmark their own performance, to improve upon current methods, and to evaluate new technologies. Additionally, we demonstrate how the yeast reference, spiked with human proteins, can be used to benchmark the power of proteomics platforms for detection of differentially expressed proteins at different levels of concentration in a complex matrix, thereby providing a metric to evaluate and minimize preanalytical and analytical variation in comparative proteomics experiments. PMID:19858499

Quantitative proteomics analysis using 2D-PAGE to investigate the effects of cigarette smoke and aerosol of a prototypic modified risk tobacco product on the lung proteome in C57BL/6 mice.

PubMed

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 the much lower effects of pMRTP aerosol than cigarette smoke on the mouse lung proteome. The combined analysis of 2D-PAGE and LC-MS/MS data identified an effect of cigarette smoke on the proteasome and actin cytoskeleton in the lung. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Facile preparation of salivary extracellular vesicles for cancer proteomics

NASA Astrophysics Data System (ADS)

Sun, Yan; Xia, Zhijun; Shang, Zhi; Sun, Kaibo; Niu, Xiaomin; Qian, Liqiang; Fan, Liu-Yin; Cao, Cheng-Xi; Xiao, Hua

2016-04-01

Extracellular vesicles (EVs) are membrane surrounded structures released by cells, which have been increasingly recognized as mediators of intercellular communication. Recent reports indicate that EVs participate in important biological processes and could serve as potential source for cancer biomarkers. As an attractive EVs source with merit of non-invasiveness, human saliva is a unique medium for clinical diagnostics. Thus, we proposed a facile approach to prepare salivary extracellular vesicles (SEVs). Affinity chromatography column combined with filter system (ACCF) was developed to efficiently remove the high abundant proteins and viscous interferences of saliva. Protein profiling in the SEVs obtained by this strategy was compared with conventional centrifugation method, which demonstrated that about 70% more SEVs proteins could be revealed. To explore its utility for cancer proteomics, we analyzed the proteome of SEVs in lung cancer patients and normal controls. Shotgun proteomic analysis illustrated that 113 and 95 proteins have been identified in cancer group and control group, respectively. Among those 63 proteins that have been consistently discovered only in cancer group, 12 proteins are lung cancer related. Our results demonstrated that SEVs prepared through the developed strategy are valuable samples for proteomics and could serve as a promising liquid biopsy for cancer.

Proteomics identifies the composition and manufacturing recipe of the 2500-year old sourdough bread from Subeixi cemetery in China.

PubMed

Shevchenko, Anna; Yang, Yimin; Knaust, Andrea; Thomas, Henrik; Jiang, Hongen; Lu, Enguo; Wang, Changsui; Shevchenko, Andrej

2014-06-13

We report on the geLC-MS/MS proteomics analysis of cereals and cereal food excavated in Subeixi cemetery (500-300BC) in Xinjiang, China. Proteomics provided direct evidence that at the Subexi sourdough bread was made from barley and broomcorn millet by leavening with a renewable starter comprising baker's yeast and lactic acid bacteria. The baking recipe and flour composition indicated that barley and millet bread belonged to the staple food already in the first millennium BC and suggested the role of Turpan basin as a major route for cultural communication between Western and Eastern Eurasia in antiquity. This article is part of a Special Issue entitled: Proteomics of non-model organisms. We demonstrate that organic residues of thousand year old foods unearthed by archeological excavations can be analyzed by geLC-MS/MS proteomics with good representation of protein source organisms and coverage of sequences of identified proteins. In-depth look into the foods proteome identifies the food type and its individual ingredients, reveals ancient food processing technologies, projects their social and economic impact and provides evidence of intercultural communication between ancient populations. Proteomics analysis of ancient organic residues is direct, quantitative and informative and therefore has the potential to develop into a valuable, generally applicable tool in archaeometry. This article is part of a Special Issue entitled: Proteomics of non-model organisms. Copyright © 2013. Published by Elsevier B.V.

Complex and extensive post-transcriptional regulation revealed by integrative proteomic and transcriptomic analysis of metabolite stress response in Clostridium acetobutylicum.

PubMed

Venkataramanan, Keerthi P; Min, Lie; Hou, Shuyu; Jones, Shawn W; Ralston, Matthew T; Lee, Kelvin H; Papoutsakis, E Terry

2015-01-01

Clostridium acetobutylicum is a model organism for both clostridial biology and solvent production. The organism is exposed to its own toxic metabolites butyrate and butanol, which trigger an adaptive stress response. Integrative analysis of proteomic and RNAseq data may provide novel insights into post-transcriptional regulation. The identified iTRAQ-based quantitative stress proteome is made up of 616 proteins with a 15 % genome coverage. The differentially expressed proteome correlated poorly with the corresponding differential RNAseq transcriptome. Up to 31 % of the differentially expressed proteins under stress displayed patterns opposite to those of the transcriptome, thus suggesting significant post-transcriptional regulation. The differential proteome of the translation machinery suggests that cells employ a different subset of ribosomal proteins under stress. Several highly upregulated proteins but with low mRNA levels possessed mRNAs with long 5'UTRs and strong RBS scores, thus supporting the argument that regulatory elements on the long 5'UTRs control their translation. For example, the oxidative stress response rubrerythrin was upregulated only at the protein level up to 40-fold without significant mRNA changes. We also identified many leaderless transcripts, several displaying different transcriptional start sites, thus suggesting mRNA-trimming mechanisms under stress. Downregulation of Rho and partner proteins pointed to changes in transcriptional elongation and termination under stress. The integrative proteomic-transcriptomic analysis demonstrated complex expression patterns of a large fraction of the proteome. Such patterns could not have been detected with one or the other omic analyses. Our analysis proposes the involvement of specific molecular mechanisms of post-transcriptional regulation to explain the observed complex stress response.

Serum proteomic analysis of extracorporeal shock wave therapy-enhanced diabetic wound healing in a streptozotocin-induced diabetes model.

PubMed

Yang, Ming-Yu; Chiang, Yuan-Cheng; Huang, Yu-Ting; Chen, Chien-Chang; Wang, Feng-Sheng; Wang, Ching-Jen; Kuo, Yur-Ren

2014-01-01

Previous studies have demonstrated that extracorporeal shock wave therapy has a significant positive effect on accelerating diabetic wound healing. However, the systemic effect after therapy is still unclear. This study investigated the plasma protein expression in the extracorporeal shock wave therapy group and diabetic controls using proteomic study. A dorsal skin defect (6 × 5 cm) in a streptozotocin-induced diabetic Wistar rat model was used. Diabetic rats receiving either no therapy or extracorporeal shock wave therapy after wounding were analyzed. The spots of interest were subjected to in-gel trypsin digestion and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to elucidate the peptide mass fingerprints. The mass spectrometric characteristics of the identified proteins, including their theoretical isoelectric points, molecular weights, sequence coverage, and Mascot score, were analyzed. Protein expression was validated using immunohistochemical analysis of topical periwounding tissues. The proteomic study revealed that at days 3 and 10 after therapy rats had significantly higher abundance of haptoglobin and significantly lower levels of the vitamin D-binding protein precursor as compared with the diabetic controls. Immunohistochemical staining of topical periwounding tissue also revealed significant upregulation of haptoglobin and downregulation of vitamin D-binding protein expression in the extracorporeal shock wave therapy group, which was consistent with the systemic proteome study. Proteome analyses demonstrated an upregulation of haptoglobin and a downregulation of vitamin D-binding protein in extracorporeal shock wave therapy-enhanced diabetic wound healing.

Application of clinical assay quality control (QC) to multivariate proteomics data: a workflow exemplified by 2-DE QC.

PubMed

Jackson, David; Bramwell, David

2013-12-16

Proteomics technologies can be effective for the discovery and assay of protein forms altered with disease. However, few examples of successful biomarker discovery yet exist. Critical to addressing this is the widespread implementation of appropriate QC (quality control) methodology. Such QC should combine the rigour of clinical laboratory assays with a suitable treatment of the complexity of the proteome by targeting separate assignable causes of variation. We demonstrate an approach, metric and example workflow for users to develop such targeted QC rules systematically and objectively, using a publicly available plasma DIGE data set. Hierarchical clustering analysis of standard channels is first used to discover correlated groups of features corresponding to specific assignable sources of technical variation. These effects are then quantified using a statistical distance metric, and followed on control charts. This allows measurement of process drift and the detection of runs that outlie for any given effect. A known technical issue on originally rejected gels was detected validating this approach, and relevant novel effects were also detected and classified effectively. Our approach was effective for 2-DE QC. Whilst we demonstrated this in a retrospective DIGE experiment, the principles would apply to ongoing QC and other proteomic technologies. This work asserts that properly carried out QC is essential to proteomics discovery experiments. Its significance is that it provides one possible novel framework for applying such methods, with a particular consideration of how to handle the complexity of the proteome. It not only focusses on 2DE-based methodology but also demonstrates general principles. A combination of results and discussion based upon a publicly available data set is used to illustrate the approach and allows a structured discussion of factors that experimenters may wish to bear in mind in other situations. The demonstration is on retrospective data only for reasons of scope, but the principles applied are also important for ongoing QC, and this work serves as a step towards a later demonstration of that application. This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics. © 2013.

Predicting Amyloidogenic Proteins in the Proteomes of Plants.

PubMed

Antonets, Kirill S; Nizhnikov, Anton A

2017-10-16

Amyloids are protein fibrils with characteristic spatial structure. Though amyloids were long perceived to be pathogens that cause dozens of incurable pathologies in humans and mammals, it is currently clear that amyloids also represent a functionally important form of protein structure implicated in a variety of biological processes in organisms ranging from archaea and bacteria to fungi and animals. Despite their social significance, plants remain the most poorly studied group of organisms in the field of amyloid biology. To date, amyloid properties have only been demonstrated in vitro or in heterologous systems for a small number of plant proteins. Here, for the first time, we performed a comprehensive analysis of the distribution of potentially amyloidogenic proteins in the proteomes of approximately 70 species of land plants using the Waltz and SARP (Sequence Analysis based on the Ranking of Probabilities) bioinformatic algorithms. We analyzed more than 2.9 million protein sequences and found that potentially amyloidogenic proteins are abundant in plant proteomes. We found that such proteins are overrepresented among membrane as well as DNA- and RNA-binding proteins of plants. Moreover, seed storage and defense proteins of most plant species are rich in amyloidogenic regions. Taken together, our data demonstrate the diversity of potentially amyloidogenic proteins in plant proteomes and suggest biological processes where formation of amyloids might be functionally important.

Comparative proteomics analysis of oral cancer cell lines: identification of cancer associated proteins

PubMed Central

2014-01-01

Background A limiting factor in performing proteomics analysis on cancerous cells is the difficulty in obtaining sufficient amounts of starting material. Cell lines can be used as a simplified model system for studying changes that accompany tumorigenesis. This study used two-dimensional gel electrophoresis (2DE) to compare the whole cell proteome of oral cancer cell lines vs normal cells in an attempt to identify cancer associated proteins. Results Three primary cell cultures of normal cells with a limited lifespan without hTERT immortalization have been successfully established. 2DE was used to compare the whole cell proteome of these cells with that of three oral cancer cell lines. Twenty four protein spots were found to have changed in abundance. MALDI TOF/TOF was then used to determine the identity of these proteins. Identified proteins were classified into seven functional categories – structural proteins, enzymes, regulatory proteins, chaperones and others. IPA core analysis predicted that 18 proteins were related to cancer with involvements in hyperplasia, metastasis, invasion, growth and tumorigenesis. The mRNA expressions of two proteins – 14-3-3 protein sigma and Stress-induced-phosphoprotein 1 – were found to correlate with the corresponding proteins’ abundance. Conclusions The outcome of this analysis demonstrated that a comparative study of whole cell proteome of cancer versus normal cell lines can be used to identify cancer associated proteins. PMID:24422745

Introducing the CPL/MUW proteome database: interpretation of human liver and liver cancer proteome profiles by referring to isolated primary cells.

PubMed

Wimmer, Helge; Gundacker, Nina C; Griss, Johannes; Haudek, Verena J; Stättner, Stefan; Mohr, Thomas; Zwickl, Hannes; Paulitschke, Verena; Baron, David M; Trittner, Wolfgang; Kubicek, Markus; Bayer, Editha; Slany, Astrid; Gerner, Christopher

2009-06-01

Interpretation of proteome data with a focus on biomarker discovery largely relies on comparative proteome analyses. Here, we introduce a database-assisted interpretation strategy based on proteome profiles of primary cells. Both 2-D-PAGE and shotgun proteomics are applied. We obtain high data concordance with these two different techniques. When applying mass analysis of tryptic spot digests from 2-D gels of cytoplasmic fractions, we typically identify several hundred proteins. Using the same protein fractions, we usually identify more than thousand proteins by shotgun proteomics. The data consistency obtained when comparing these independent data sets exceeds 99% of the proteins identified in the 2-D gels. Many characteristic differences in protein expression of different cells can thus be independently confirmed. Our self-designed SQL database (CPL/MUW - database of the Clinical Proteomics Laboratories at the Medical University of Vienna accessible via www.meduniwien.ac.at/proteomics/database) facilitates (i) quality management of protein identification data, which are based on MS, (ii) the detection of cell type-specific proteins and (iii) of molecular signatures of specific functional cell states. Here, we demonstrate, how the interpretation of proteome profiles obtained from human liver tissue and hepatocellular carcinoma tissue is assisted by the Clinical Proteomics Laboratories at the Medical University of Vienna-database. Therefore, we suggest that the use of reference experiments supported by a tailored database may substantially facilitate data interpretation of proteome profiling experiments.

COMPASS: a suite of pre- and post-search proteomics software tools for OMSSA

PubMed Central

Wenger, Craig D.; Phanstiel, Douglas H.; Lee, M. Violet; Bailey, Derek J.; Coon, Joshua J.

2011-01-01

Here we present the Coon OMSSA Proteomic Analysis Software Suite (COMPASS): a free and open-source software pipeline for high-throughput analysis of proteomics data, designed around the Open Mass Spectrometry Search Algorithm. We detail a synergistic set of tools for protein database generation, spectral reduction, peptide false discovery rate analysis, peptide quantitation via isobaric labeling, protein parsimony and protein false discovery rate analysis, and protein quantitation. We strive for maximum ease of use, utilizing graphical user interfaces and working with data files in the original instrument vendor format. Results are stored in plain text comma-separated values files, which are easy to view and manipulate with a text editor or spreadsheet program. We illustrate the operation and efficacy of COMPASS through the use of two LC–MS/MS datasets. The first is a dataset of a highly annotated mixture of standard proteins and manually validated contaminants that exhibits the identification workflow. The second is a dataset of yeast peptides, labeled with isobaric stable isotope tags and mixed in known ratios, to demonstrate the quantitative workflow. For these two datasets, COMPASS performs equivalently or better than the current de facto standard, the Trans-Proteomic Pipeline. PMID:21298793

Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics.

PubMed

Garnett, Graham A E; Starke, Melissa J; Shaurya, Alok; Li, Janessa; Hof, Fraser

2016-04-05

Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods.

The proteomic landscape of triple-negative breast cancer.

PubMed

Lawrence, Robert T; Perez, Elizabeth M; Hernández, Daniel; Miller, Chris P; Haas, Kelsey M; Irie, Hanna Y; Lee, Su-In; Blau, C Anthony; Villén, Judit

2015-04-28

Triple-negative breast cancer is a heterogeneous disease characterized by poor clinical outcomes and a shortage of targeted treatment options. To discover molecular features of triple-negative breast cancer, we performed quantitative proteomics analysis of twenty human-derived breast cell lines and four primary breast tumors to a depth of more than 12,000 distinct proteins. We used this data to identify breast cancer subtypes at the protein level and demonstrate the precise quantification of biomarkers, signaling proteins, and biological pathways by mass spectrometry. We integrated proteomics data with exome sequence resources to identify genomic aberrations that affect protein expression. We performed a high-throughput drug screen to identify protein markers of drug sensitivity and understand the mechanisms of drug resistance. The genome and proteome provide complementary information that, when combined, yield a powerful engine for therapeutic discovery. This resource is available to the cancer research community to catalyze further analysis and investigation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

A proteomic landscape of diffuse-type gastric cancer.

PubMed

Ge, Sai; Xia, Xia; Ding, Chen; Zhen, Bei; Zhou, Quan; Feng, Jinwen; Yuan, Jiajia; Chen, Rui; Li, Yumei; Ge, Zhongqi; Ji, Jiafu; Zhang, Lianhai; Wang, Jiayuan; Li, Zhongwu; Lai, Yumei; Hu, Ying; Li, Yanyan; Li, Yilin; Gao, Jing; Chen, Lin; Xu, Jianming; Zhang, Chunchao; Jung, Sung Yun; Choi, Jong Min; Jain, Antrix; Liu, Mingwei; Song, Lei; Liu, Wanlin; Guo, Gaigai; Gong, Tongqing; Huang, Yin; Qiu, Yang; Huang, Wenwen; Shi, Tieliu; Zhu, Weimin; Wang, Yi; He, Fuchu; Shen, Lin; Qin, Jun

2018-03-08

The diffuse-type gastric cancer (DGC) is a subtype of gastric cancer with the worst prognosis and few treatment options. Here we present a dataset from 84 DGC patients, composed of a proteome of 11,340 gene products and mutation information of 274 cancer driver genes covering paired tumor and nearby tissue. DGC can be classified into three subtypes (PX1-3) based on the altered proteome alone. PX1 and PX2 exhibit dysregulation in the cell cycle and PX2 features an additional EMT process; PX3 is enriched in immune response proteins, has the worst survival, and is insensitive to chemotherapy. Data analysis revealed four major vulnerabilities in DGC that may be targeted for treatment, and allowed the nomination of potential immunotherapy targets for DGC patients, particularly for those in PX3. This dataset provides a rich resource for information and knowledge mining toward altered signaling pathways in DGC and demonstrates the benefit of proteomic analysis in cancer molecular subtyping.

Proteomic characterization of the outer membrane vesicle of the halophilic marine bacterium Novosphingobium pentaromativorans US6-1.

PubMed

Yun, Sung Ho; Lee, Sang-Yeop; Choi, Chi-Won; Lee, Hayoung; Ro, Hyun-Joo; Jun, Sangmi; Kwon, Yong Min; Kwon, Kae Kyoung; Kim, Sang-Jin; Kim, Gun-Hwa; Kim, Seung Il

2017-01-01

Novosphingobium pentaromativorans US6-1 is a Gram-negative halophilic marine bacterium able to utilize several polycyclic aromatic hydrocarbons such as phenanthrene, pyrene, and benzo[a]pyrene. In this study, using transmission electron microscopy, we confirmed that N. pentaromativorans US6-1 produces outer membrane vesicles (OMVs). N. pentaromativorans OMVs (hereafter OMV Novo ) are spherical in shape, and the average diameter of OMV Novo is 25-70 nm. Proteomic analysis revealed that outer membrane proteins and periplasmic proteins of N. pentaromativorans are the major protein components of OMV Novo . Comparative proteomic analysis with the membrane-associated protein fraction and correlation analysis demonstrated that the outer membrane proteins of OMV Novo originated from the membrane- associated protein fraction. To the best of our knowledge, this study is the first to characterize OMV purified from halophilic marine bacteria.

Automated Big Data Analysis in Bottom-up and Targeted Proteomics

PubMed Central

van der Plas-Duivesteijn, Suzanne; Domański, Dominik; Smith, Derek; Borchers, Christoph; Palmblad, Magnus; Mohamme, Yassene

2014-01-01

Similar to other data intensive sciences, analyzing mass spectrometry-based proteomics data involves multiple steps and diverse software using different algorithms and data formats and sizes. Besides that the distributed and evolving nature of the data in online repositories, another challenge is that a scientists have to deal with many steps of analysis pipelines. A documented data processing is also becoming an essential part for the overall reproducibility of the results. Thanks to different e-Science initiatives, scientific workflow engines have become a means for automated, sharable and reproducible data processing. While these are designed as general tools, they can be employed to solve different challenges that we are facing in handling our Big Data. Here we present three use cases: improving the performance of different spectral search engines by decomposing input data and recomposing the resulting files, building spectral libraries from more than 20 million spectra, and integrating information from multiple resources to select most appropriate peptides for targeted proteomics analyses. The three use cases demonstrate different challenges in exploiting proteomics data analysis. In the first we integrate local and cloud processing resources in order to obtain better performance resulting in more than 30-fold speed improvement. By considering search engines as legacy software our solution is applicable to multiple search algorithms. The second use case is an example of automated processing of many data files of different sizes and locations, starting with raw data and ending with the final, ready-to-use library. This demonstrates the robustness and fault tolerance when dealing with huge amount data stored in multiple files. The third use case demonstrates retrieval and integration of information and data from multiple online repositories. In addition to the diversity of data formats and Web interfaces, this use case also illustrates how to deal with incomplete data.

Urine Sample Preparation in 96-Well Filter Plates for Quantitative Clinical Proteomics

PubMed Central

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

Missing Value Monitoring Enhances the Robustness in Proteomics Quantitation.

PubMed

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.

The Cytotoxicity Mechanism of 6-Shogaol-Treated HeLa Human Cervical Cancer Cells Revealed by Label-Free Shotgun Proteomics and Bioinformatics Analysis.

PubMed

Liu, Qun; Peng, Yong-Bo; Qi, Lian-Wen; Cheng, Xiao-Lan; Xu, Xiao-Jun; Liu, Le-Le; Liu, E-Hu; Li, Ping

2012-01-01

Cervical cancer is one of the most common cancers among women in the world. 6-Shogaol is a natural compound isolated from the rhizome of ginger (Zingiber officinale). In this paper, we demonstrated that 6-shogaol induced apoptosis and G2/M phase arrest in human cervical cancer HeLa cells. Endoplasmic reticulum stress and mitochondrial pathway were involved in 6-shogaol-mediated apoptosis. Proteomic analysis based on label-free strategy by liquid chromatography chip quadrupole time-of-flight mass spectrometry was subsequently proposed to identify, in a non-target-biased manner, the molecular changes in cellular proteins in response to 6-shogaol treatment. A total of 287 proteins were differentially expressed in response to 24 h treatment with 15 μM 6-shogaol in HeLa cells. Significantly changed proteins were subjected to functional pathway analysis by multiple analyzing software. Ingenuity pathway analysis (IPA) suggested that 14-3-3 signaling is a predominant canonical pathway involved in networks which may be significantly associated with the process of apoptosis and G2/M cell cycle arrest induced by 6-shogaol. In conclusion, this work developed an unbiased protein analysis strategy by shotgun proteomics and bioinformatics analysis. Data observed provide a comprehensive analysis of the 6-shogaol-treated HeLa cell proteome and reveal protein alterations that are associated with its anticancer mechanism.

The Cytotoxicity Mechanism of 6-Shogaol-Treated HeLa Human Cervical Cancer Cells Revealed by Label-Free Shotgun Proteomics and Bioinformatics Analysis

PubMed Central

Liu, Qun; Peng, Yong-Bo; Qi, Lian-Wen; Cheng, Xiao-Lan; Xu, Xiao-Jun; Liu, Le-Le; Liu, E-Hu; Li, Ping

2012-01-01

Cervical cancer is one of the most common cancers among women in the world. 6-Shogaol is a natural compound isolated from the rhizome of ginger (Zingiber officinale). In this paper, we demonstrated that 6-shogaol induced apoptosis and G2/M phase arrest in human cervical cancer HeLa cells. Endoplasmic reticulum stress and mitochondrial pathway were involved in 6-shogaol-mediated apoptosis. Proteomic analysis based on label-free strategy by liquid chromatography chip quadrupole time-of-flight mass spectrometry was subsequently proposed to identify, in a non-target-biased manner, the molecular changes in cellular proteins in response to 6-shogaol treatment. A total of 287 proteins were differentially expressed in response to 24 h treatment with 15 μM 6-shogaol in HeLa cells. Significantly changed proteins were subjected to functional pathway analysis by multiple analyzing software. Ingenuity pathway analysis (IPA) suggested that 14-3-3 signaling is a predominant canonical pathway involved in networks which may be significantly associated with the process of apoptosis and G2/M cell cycle arrest induced by 6-shogaol. In conclusion, this work developed an unbiased protein analysis strategy by shotgun proteomics and bioinformatics analysis. Data observed provide a comprehensive analysis of the 6-shogaol-treated HeLa cell proteome and reveal protein alterations that are associated with its anticancer mechanism. PMID:23243437

Evaluation of empirical rule of linearly correlated peptide selection (ERLPS) for proteotypic peptide-based quantitative proteomics.

PubMed

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.

Assessment of SRM, MRM(3) , and DIA for the targeted analysis of phosphorylation dynamics in non-small cell lung cancer.

PubMed

Schmidlin, Thierry; Garrigues, Luc; Lane, Catherine S; Mulder, T Celine; van Doorn, Sander; Post, Harm; de Graaf, Erik L; Lemeer, Simone; Heck, Albert J R; Altelaar, A F Maarten

2016-08-01

Hypothesis-driven MS-based targeted proteomics has gained great popularity in a relatively short timespan. Next to the widely established selected reaction monitoring (SRM) workflow, data-independent acquisition (DIA), also referred to as sequential window acquisition of all theoretical spectra (SWATH) was introduced as a high-throughput targeted proteomics method. DIA facilitates increased proteome coverage, however, does not yet reach the sensitivity obtained with SRM. Therefore, a well-informed method selection is crucial for designing a successful targeted proteomics experiment. This is especially the case when targeting less conventional peptides such as those that contain PTMs, as these peptides do not always adhere to the optimal fragmentation considerations for targeted assays. Here, we provide insight into the performance of DIA, SRM, and MRM cubed (MRM(3) ) in the analysis of phosphorylation dynamics throughout the phosphoinositide 3-kinase mechanistic target of rapamycin (PI3K-mTOR) and mitogen-activated protein kinase (MAPK) signaling network. We observe indeed that DIA is less sensitive when compared to SRM, however demonstrates increased flexibility, by postanalysis selection of alternative phosphopeptide precursors. Additionally, we demonstrate the added benefit of MRM(3) , allowing the quantification of two poorly accessible phosphosites. In total, targeted proteomics enabled the quantification of 42 PI3K-mTOR and MAPK phosphosites, gaining a so far unachieved in-depth view mTOR signaling events linked to tyrosine kinase inhibitor resistance in non-small cell lung cancer. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mitochondrial proteome remodelling in pressure overload-induced heart failure: the role of mitochondrial oxidative stress

PubMed Central

Dai, Dao-Fu; Hsieh, Edward J.; Liu, Yonggang; Chen, Tony; Beyer, Richard P.; Chin, Michael T.; MacCoss, Michael J.; Rabinovitch, Peter S.

2012-01-01

Aims We investigate the role of mitochondrial oxidative stress in mitochondrial proteome remodelling using mouse models of heart failure induced by pressure overload. Methods and results We demonstrate that mice overexpressing catalase targeted to mitochondria (mCAT) attenuate pressure overload-induced heart failure. An improved method of label-free unbiased analysis of the mitochondrial proteome was applied to the mouse model of heart failure induced by transverse aortic constriction (TAC). A total of 425 mitochondrial proteins were compared between wild-type and mCAT mice receiving TAC or sham surgery. The changes in the mitochondrial proteome in heart failure included decreased abundance of proteins involved in fatty acid metabolism, an increased abundance of proteins in glycolysis, apoptosis, mitochondrial unfolded protein response and proteolysis, transcription and translational control, and developmental processes as well as responses to stimuli. Overexpression of mCAT better preserved proteins involved in fatty acid metabolism and attenuated the increases in apoptotic and proteolytic enzymes. Interestingly, gene ontology analysis also showed that monosaccharide metabolic processes and protein folding/proteolysis were only overrepresented in mCAT but not in wild-type mice in response to TAC. Conclusion This is the first study to demonstrate that scavenging mitochondrial reactive oxygen species (ROS) by mCAT not only attenuates most of the mitochondrial proteome changes in heart failure, but also induces a subset of unique alterations. These changes represent processes that are adaptive to the increased work and metabolic requirements of pressure overload, but which are normally inhibited by overproduction of mitochondrial ROS. PMID:22012956

Isolation and Proteomic Characterization of the Arabidopsis Golgi Defines Functional and Novel Components Involved in Plant Cell Wall Biosynthesis1[W][OA

PubMed Central

Parsons, Harriet T.; Christiansen, Katy; Knierim, Bernhard; Carroll, Andrew; Ito, Jun; Batth, Tanveer S.; Smith-Moritz, Andreia M.; Morrison, Stephanie; McInerney, Peter; Hadi, Masood Z.; Auer, Manfred; Mukhopadhyay, Aindrila; Petzold, Christopher J.; Scheller, Henrik V.; Loqué, Dominique; Heazlewood, Joshua L.

2012-01-01

The plant Golgi plays a pivotal role in the biosynthesis of cell wall matrix polysaccharides, protein glycosylation, and vesicle trafficking. Golgi-localized proteins have become prospective targets for reengineering cell wall biosynthetic pathways for the efficient production of biofuels from plant cell walls. However, proteomic characterization of the Golgi has so far been limited, owing to the technical challenges inherent in Golgi purification. In this study, a combination of density centrifugation and surface charge separation techniques have allowed the reproducible isolation of Golgi membranes from Arabidopsis (Arabidopsis thaliana) at sufficiently high purity levels for in-depth proteomic analysis. Quantitative proteomic analysis, immunoblotting, enzyme activity assays, and electron microscopy all confirm high purity levels. A composition analysis indicated that approximately 19% of proteins were likely derived from contaminating compartments and ribosomes. The localization of 13 newly assigned proteins to the Golgi using transient fluorescent markers further validated the proteome. A collection of 371 proteins consistently identified in all replicates has been proposed to represent the Golgi proteome, marking an appreciable advancement in numbers of Golgi-localized proteins. A significant proportion of proteins likely involved in matrix polysaccharide biosynthesis were identified. The potential within this proteome for advances in understanding Golgi processes has been demonstrated by the identification and functional characterization of the first plant Golgi-resident nucleoside diphosphatase, using a yeast complementation assay. Overall, these data show key proteins involved in primary cell wall synthesis and include a mixture of well-characterized and unknown proteins whose biological roles and importance as targets for future research can now be realized. PMID:22430844

A tutorial for software development in quantitative proteomics using PSI standard formats☆

PubMed Central

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

Completed | Office of Cancer Clinical Proteomics Research

Cancer.gov

Prior to the current Clinical Proteomic Tumor Analysis Consortium (CPTAC), previously funded initiatives associated with clinical proteomics research included: Clinical Proteomic Tumor Analysis Consortium (CPTAC 2.0) Clinical Proteomic Technologies for Cancer Initiative (CPTC) Mouse Proteomic Technologies Initiative

A-to-I RNA Editing Contributes to Proteomic Diversity in Cancer.

PubMed

Peng, Xinxin; Xu, Xiaoyan; Wang, Yumeng; Hawke, David H; Yu, Shuangxing; Han, Leng; Zhou, Zhicheng; Mojumdar, Kamalika; Jeong, Kang Jin; Labrie, Marilyne; Tsang, Yiu Huen; Zhang, Minying; Lu, Yiling; Hwu, Patrick; Scott, Kenneth L; Liang, Han; Mills, Gordon B

2018-05-14

Adenosine (A) to inosine (I) RNA editing introduces many nucleotide changes in cancer transcriptomes. However, due to the complexity of post-transcriptional regulation, the contribution of RNA editing to proteomic diversity in human cancers remains unclear. Here, we performed an integrated analysis of TCGA genomic data and CPTAC proteomic data. Despite limited site diversity, we demonstrate that A-to-I RNA editing contributes to proteomic diversity in breast cancer through changes in amino acid sequences. We validate the presence of editing events at both RNA and protein levels. The edited COPA protein increases proliferation, migration, and invasion of cancer cells in vitro. Our study suggests an important contribution of A-to-I RNA editing to protein diversity in cancer and highlights its translational potential. Copyright © 2018 Elsevier Inc. All rights reserved.

The State of the Human Proteome in 2013 as viewed through PeptideAtlas: Comparing the Kidney, Urine, and Plasma Proteomes for the Biology and Disease-driven Human Proteome Project

PubMed Central

Farrah, Terry; Deutsch, Eric W.; Omenn, Gilbert S.; Sun, Zhi; Watts, Julian D.; Yamamoto, Tadashi; Shteynberg, David; Harris, Micheleen M.; Moritz, Robert L.

2014-01-01

The kidney, urine, and plasma proteomes are intimately related: proteins and metabolic waste products are filtered from the plasma by the kidney and excreted via the urine, while kidney proteins may be secreted into the circulation or released into the urine. Shotgun proteomics datasets derived from human kidney, urine, and plasma samples were collated and processed using a uniform software pipeline, and relative protein abundances were estimated by spectral counting. The resulting PeptideAtlas builds yielded 4005, 2491, and 3553 nonredundant proteins at 1% FDR for the kidney, urine, and plasma proteomes, respectively—for kidney and plasma, the largest high-confidence protein sets to date. The same pipeline applied to all available human data yielded a 2013 Human PeptideAtlas build containing 12,644 nonredundant proteins and at least one peptide for each of ~14,000 Swiss-Prot entries, an increase over 2012 of ~7.5% of the predicted human proteome. We demonstrate that abundances are correlated between plasma and urine, examine the most abundant urine proteins not derived from either plasma or kidney, and consider the biomarker potential of proteins associated with renal decline. This analysis forms part of the Biology and Disease-driven Human Proteome Project (B/D-HPP) and a contribution to the Chromosome-centric Human Proteome Project (C-HPP) special issue. PMID:24261998

Two independent proteomic approaches provide a comprehensive analysis of the synovial fluid proteome response to Autologous Chondrocyte Implantation.

PubMed

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 success or failure. Functional pathways that are dysregulated in ACI non-responders were identified, including acute-phase response signalling. Several candidate biomarkers for baseline prediction of ACI outcome were identified. A holistic overview of the SF proteome in responders and non-responders to ACI  has been profiled, providing a better understanding of the biological pathways underlying clinical outcome, particularly the differential response to cartilage harvest in non-responders.

Improved metabolites of pharmaceutical ingredient grade Ginkgo biloba and the correlated proteomics analysis.

PubMed

Zheng, Wen; Li, Ximin; Zhang, Lin; Zhang, Yanzhen; Lu, Xiaoping; Tian, Jingkui

2015-06-01

Ginkgo biloba is an attractive and traditional medicinal plant, and has been widely used as a phytomedicine in the prevention and treatment of cardiovascular and cerebrovascular diseases. Flavonoids and terpene lactones are the major bioactive components of Ginkgo, whereas the ginkgolic acids (GAs) with strong allergenic properties are strictly controlled. In this study, we tested the content of flavonoids and GAs under ultraviolet-B (UV-B) treatment and performed comparative proteomic analyses to determine the differential proteins that occur upon UV-B radiation. That might play a crucial role in producing flavonoids and GAs. Our phytochemical analyses demonstrated that UV-B irradiation significantly increased the content of active flavonoids, and decreased the content of toxic GAs. We conducted comparative proteomic analysis of both whole leaf and chloroplasts proteins. In total, 27 differential proteins in the whole leaf and 43 differential proteins in the chloroplast were positively identified and functionally annotated. The proteomic data suggested that enhanced UV-B radiation exposure activated antioxidants and stress-responsive proteins as well as reduced the rate of photosynthesis. We demonstrate that UV-B irradiation pharmaceutically improved the metabolic ingredients of Ginkgo, particularly in terms of reducing GAs. With high UV absorption properties, and antioxidant activities, the flavonoids were likely highly induced as protective molecules following UV-B irradiation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomics Analysis of Skeletal Muscle from Leptin-Deficient ob/ob Mice Reveals Adaptive Remodeling of Metabolic Characteristics and Fiber Type Composition.

PubMed

Schönke, Milena; Björnholm, Marie; Chibalin, Alexander V; Zierath, Juleen R; Deshmukh, Atul S

2018-03-01

Skeletal muscle insulin resistance, an early metabolic defect in the pathogenesis of type 2 diabetes (T2D), may be a cause or consequence of altered protein expression profiles. Proteomics technology offers enormous promise to investigate molecular mechanisms underlying pathologies, however, the analysis of skeletal muscle is challenging. Using state-of-the-art multienzyme digestion and filter-aided sample preparation (MED-FASP) and a mass spectrometry (MS)-based workflow, we performed a global proteomics analysis of skeletal muscle from leptin-deficient, obese, insulin resistant (ob/ob) and lean mice in mere two fractions in a short time (8 h per sample). We identified more than 6000 proteins with 118 proteins differentially regulated in obesity. This included protein kinases, phosphatases, and secreted and fiber type associated proteins. Enzymes involved in lipid metabolism in skeletal muscle from ob/ob mice were increased, providing evidence against reduced fatty acid oxidation in lipid-induced insulin resistance. Mitochondrial and peroxisomal proteins, as well as components of pyruvate and lactate metabolism, were increased. Finally, the skeletal muscle proteome from ob/ob mice displayed a shift toward the "slow fiber type." This detailed characterization of an obese rodent model of T2D demonstrates an efficient workflow for skeletal muscle proteomics, which may easily be adapted to other complex tissues. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Combined analysis of transcriptome and proteome data as a tool for the identification of candidate biomarkers in renal cell carcinoma

PubMed Central

Seliger, Barbara; Dressler, Sven P.; Wang, Ena; Kellner, Roland; Recktenwald, Christian V.; Lottspeich, Friedrich; Marincola, Francesco M.; Baumgärtner, Maja; Atkins, Derek; Lichtenfels, Rudolf

2012-01-01

Results obtained from expression profilings of renal cell carcinoma using different “ome”-based approaches and comprehensive data analysis demonstrated that proteome-based technologies and cDNA microarray analyses complement each other during the discovery phase for disease-related candidate biomarkers. The integration of the respective data revealed the uniqueness and complementarities of the different technologies. While comparative cDNA microarray analyses though restricted to upregulated targets largely revealed genes involved in controlling gene/protein expression (19%) and signal transduction processes (13%), proteomics/PROTEOMEX-defined candidate biomarkers include enzymes of the cellular metabolism (36%), transport proteins (12%) and cell motility/structural molecules (10%). Candidate biomarkers defined by proteomics and PROTEOMEX are frequently shared, whereas the sharing rate between cDNA microarray and proteome-based profilings is limited. Putative candidate biomarkers provide insights into their cellular (dys)function and their diagnostic/prognostic value but still warrant further validation in larger patient numbers. Based on the fact that merely 3 candidate biomarkers were shared by all applied technologies, namely annexin A4, tubulin alpha-1A chain and ubiquitin carboxyl-terminal hydrolase L1 the analysis at a single hierarchical level of biological regulation seems to provide only limited results thus emphasizing the importance and benefit of performing rather combinatorial screenings which can complement the standard clinical predictors. PMID:19235166

Proteomic identification of processes and pathways characteristic of osmoregulatory tissues in spiny dogfish shark (Squalus acanthias).

PubMed

Lee, Jinoo; Valkova, Nelly; White, Mark P; Kültz, Dietmar

2006-09-01

We used dogfish shark (Squalus acanthias) as a model for proteome analysis of six different tissues to evaluate tissue-specific protein expression on a global scale and to deduce specific functions and the relatedness of multiple tissues from their proteomes. Proteomes of heart, brain, kidney, intestine, gill, and rectal gland were separated by two-dimensional gel electrophoresis (2DGE), gel images were matched using Delta 2D software and then evaluated for tissue-specific proteins. Sixty-one proteins (4%) were found to be in only a single type of tissue and 535 proteins (36%) were equally abundant in all six tissues. Relatedness between tissues was assessed based on tissue-specific expression patterns of all 1465 consistently resolved protein spots. This analysis revealed that tissues with osmoregulatory function (kidney, intestine, gill, rectal gland) were more similar in their overall proteomes than non-osmoregulatory tissues (heart, brain). Sixty-one proteins were identified by MALDI-TOF/TOF mass spectrometry and biological functions characteristic of osmoregulatory tissues were derived from gene ontology and molecular pathway analysis. Our data demonstrate that the molecular machinery for energy and urea metabolism and the Rho-GTPase/cytoskeleton pathway are enriched in osmoregulatory tissues of sharks. Our work provides a strong rationale for further study of the contribution of these mechanisms to the osmoregulation of marine sharks.

Proteomics-based network analysis characterizes biological processes and pathways activated by preconditioned mesenchymal stem cells in cardiac repair mechanisms.

PubMed

Di Silvestre, Dario; Brambilla, Francesca; Scardoni, Giovanni; Brunetti, Pietro; Motta, Sara; Matteucci, Marco; Laudanna, Carlo; Recchia, Fabio A; Lionetti, Vincenzo; Mauri, Pierluigi

2017-05-01

We have demonstrated that intramyocardial delivery of human mesenchymal stem cells preconditioned with a hyaluronan mixed ester of butyric and retinoic acid (MSCp + ) is more effective in preventing the decay of regional myocardial contractility in a swine model of myocardial infarction (MI). However, the understanding of the role of MSCp + in proteomic remodeling of cardiac infarcted tissue is not complete. We therefore sought to perform a comprehensive analysis of the proteome of infarct remote (RZ) and border zone (BZ) of pigs treated with MSCp + or unconditioned stem cells. Heart tissues were analyzed by MudPIT and differentially expressed proteins were selected by a label-free approach based on spectral counting. Protein profiles were evaluated by using PPI networks and their topological analysis. The proteomic remodeling was largely prevented in MSCp + group. Extracellular proteins involved in fibrosis were down-regulated, while energetic pathways were globally up-regulated. Cardioprotectant pathways involved in the production of keto acid metabolites were also activated. Additionally, we found that new hub proteins support the cardioprotective phenotype characterizing the left ventricular BZ treated with MSCp + . In fact, the up-regulation of angiogenic proteins NCL and RAC1 can be explained by the increase of capillary density induced by MSCp + . Our results show that angiogenic pathways appear to be uniquely positioned to integrate signaling with energetic pathways involving cardiac repair. Our findings prompt the use of proteomics-based network analysis to optimize new approaches preventing the post-ischemic proteomic remodeling that may underlie the limited self-repair ability of adult heart. Copyright © 2017 Elsevier B.V. All rights reserved.

iProphet: Multi-level Integrative Analysis of Shotgun Proteomic Data Improves Peptide and Protein Identification Rates and Error Estimates*

PubMed Central

Shteynberg, David; Deutsch, Eric W.; Lam, Henry; Eng, Jimmy K.; Sun, Zhi; Tasman, Natalie; Mendoza, Luis; Moritz, Robert L.; Aebersold, Ruedi; Nesvizhskii, Alexey I.

2011-01-01

The combination of tandem mass spectrometry and sequence database searching is the method of choice for the identification of peptides and the mapping of proteomes. Over the last several years, the volume of data generated in proteomic studies has increased dramatically, which challenges the computational approaches previously developed for these data. Furthermore, a multitude of search engines have been developed that identify different, overlapping subsets of the sample peptides from a particular set of tandem mass spectrometry spectra. We present iProphet, the new addition to the widely used open-source suite of proteomic data analysis tools Trans-Proteomics Pipeline. Applied in tandem with PeptideProphet, it provides more accurate representation of the multilevel nature of shotgun proteomic data. iProphet combines the evidence from multiple identifications of the same peptide sequences across different spectra, experiments, precursor ion charge states, and modified states. It also allows accurate and effective integration of the results from multiple database search engines applied to the same data. The use of iProphet in the Trans-Proteomics Pipeline increases the number of correctly identified peptides at a constant false discovery rate as compared with both PeptideProphet and another state-of-the-art tool Percolator. As the main outcome, iProphet permits the calculation of accurate posterior probabilities and false discovery rate estimates at the level of sequence identical peptide identifications, which in turn leads to more accurate probability estimates at the protein level. Fully integrated with the Trans-Proteomics Pipeline, it supports all commonly used MS instruments, search engines, and computer platforms. The performance of iProphet is demonstrated on two publicly available data sets: data from a human whole cell lysate proteome profiling experiment representative of typical proteomic data sets, and from a set of Streptococcus pyogenes experiments more representative of organism-specific composite data sets. PMID:21876204

Top-down proteomics reveals a unique protein S-thiolation switch in Salmonella Typimurium in response to infection-like conditions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ansong, Charles; Wu, Si; Meng, Da

Characterization of the mature protein complement in cells is crucial for a better understanding of cellular processes on a systems-wide scale. Bottom-up proteomic approaches often lead to loss of critical information about an endogenous protein’s actual state due to post translational modifications (PTMs) and other processes. Top-down approaches that involve analysis of the intact protein can address this concern but present significant analytical challenges related to the separation quality needed, measurement sensitivity, and speed that result in low throughput and limited coverage. Here we used single-dimension ultra high pressure liquid chromatography mass spectrometry to investigate the comprehensive ‘intact’ proteome ofmore » the Gram negative bacterial pathogen Salmonella Typhimurium. Top-down proteomics analysis revealed 563 unique proteins including 1665 proteoforms generated by PTMs, representing the largest microbial top-down dataset reported to date. Our analysis not only confirmed several previously recognized aspects of Salmonella biology and bacterial PTMs in general, but also revealed several novel biological insights. Of particular interest was differential utilization of the protein S-thiolation forms S-glutathionylation and S-cysteinylation in response to infection-like conditions versus basal conditions, which was corroborated by changes in corresponding biosynthetic pathways. This differential utilization highlights underlying metabolic mechanisms that modulate changes in cellular signaling, and represents to our knowledge the first report of S-cysteinylation in Gram negative bacteria. The demonstrated utility of our simple proteome-wide intact protein level measurement strategy for gaining biological insight should promote broader adoption and applications of top-down proteomics approaches.« less

Lipoaspirate fluid proteome: A preliminary investigation by LC-MS top-down/bottom-up integrated platform of a high potential biofluid in regenerative medicine.

PubMed

Inserra, Ilaria; Martelli, Claudia; Cipollina, Mara; Cicione, Claudia; Iavarone, Federica; Taranto, Giuseppe Di; Barba, Marta; Castagnola, Massimo; Desiderio, Claudia; Lattanzi, Wanda

2016-04-01

The lipoaspirate fluid (LAF) is emerging as a potentially valuable source in regenerative medicine. In particular, our group recently demonstrated that it is able to exert osteoinductive properties in vitro. This original observation stimulated the investigation of the proteomic component of LAF, by means of LC-ESI-LTQ-Orbitrap-MS top-down/bottom-up integrated approach, which represents the object of the present study. Top-down analyses required the optimization of sample pretreatment procedures to enable the correct investigation of the intact proteome. Bottom-up analyses have been directly applied to untreated samples after monodimensional SDS-PAGE separation. The analysis of the acid-soluble fraction of LAF by top-down approach allowed demonstrating the presence of albumin and hemoglobin fragments (i.e. VV- and LVV-hemorphin-7), thymosins β4 and β10 peptides, ubiquitin and acyl-CoA binding protein; adipogenesis regulatory factor, perilipin-1 fragments, and S100A6, along with their PTMs. Part of the bottom-up proteomic profile was reproducibly found in both tested samples. The bottom-up approach allowed demonstrating the presence of proteins, listed among the components of adipose tissue and/or comprised within the ASCs intracellular content and secreted proteome. Our data provide a first glance on the LAF molecular profile, which is consistent with its tissue environment. LAF appeared to contain bioactive proteins, peptides and paracrine factors, suggesting its potential translational exploitation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inactivation of West Nile Virus in Serum with Heat, Ionic Detergent, and Reducing Agent for Proteomic Applications (Open Access Publisher’s Version)

DTIC Science & Technology

2017-05-19

LightCycler® 96 desktop software. Positive and negative samples were identified using the “ Qualitative Detection” analysis function using the default...Institute of Infectious Diseases, Fort Detrick, MD 21702, United States A R T I C L E I N F O Keywords: West Nile virus Virus inactivation Sample buffer... samples using a commercially available SDS- PAGE sample buffer for proteomic studies. Using this method, we demonstrate its utility by identification

Molecular diversity of toxic components from the scorpion Heterometrus petersii venom revealed by proteomic and transcriptome analysis.

PubMed

Ma, Yibao; Zhao, Yong; Zhao, Ruiming; Zhang, Weiping; He, Yawen; Wu, Yingliang; Cao, Zhijian; Guo, Lin; Li, Wenxin

2010-07-01

Scorpion venoms contain a vast untapped reservoir of natural products, which have the potential for medicinal value in drug discovery. In this study, toxin components from the scorpion Heterometrus petersii venom were evaluated by transcriptome and proteome analysis.Ten known families of venom peptides and proteins were identified, which include: two families of potassium channel toxins, four families of antimicrobial and cytolytic peptides,and one family from each of the calcium channel toxins, La1-like peptides, phospholipase A2,and the serine proteases. In addition, we also identified 12 atypical families, which include the acid phosphatases, diuretic peptides, and ten orphan families. From the data presented here, the extreme diversity and convergence of toxic components in scorpion venom was uncovered. Our work demonstrates the power of combining transcriptomic and proteomic approaches in the study of animal venoms.

Novel phage group infecting Lactobacillus delbrueckii subsp. lactis, as revealed by genomic and proteomic analysis of bacteriophage Ldl1.

PubMed

Casey, Eoghan; Mahony, Jennifer; Neve, Horst; Noben, Jean-Paul; Dal Bello, Fabio; van Sinderen, Douwe

2015-02-01

Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 +/- 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.

'Gate effect' in templated polyacrylamide membranes influences the electrotransport of proteins and finds applications in proteome analysis.

PubMed

Bossi, Alessandra; Andreoli, Matteo; Bonini, Francesca; Piletsky, Sergey

2007-09-01

Templating is an effective way for the structural modifications of a material and hence for altering its functional properties. Here protein imprinting was exploited to alter polymeric polyacrylamide (PAA) membranes. The sieving properties and selection abilities of the material formed were evaluated by studying the electrically driven transport of various proteins across templated PAA membranes. The sieving properties correlated with the templating process and depended on the quantity of template used during the polymerisation. For 1 mg/mL protein-templated membranes a 'gate effect' was shown, which induced a preferential migration of the template and of similar-size proteins. Such template preferential electrotransport was exploited for the selective removal of certain proteins in biological fluids prior to proteome analysis (depletion of albumin from human serum); the efficiency of the removal was demonstrated by analysing the serum proteome by two-dimensional electrophoresis experiments.

Proteomics analysis suggests broad functional changes in potato leaves triggered by phosphites and a complex indirect mode of action against Phytophthora infestans.

PubMed

Lim, Sanghyun; Borza, Tudor; Peters, Rick D; Coffin, Robert H; Al-Mughrabi, Khalil I; Pinto, Devanand M; Wang-Pruski, Gefu

2013-11-20

Phosphite (salts of phosphorous acid; Phi)-based fungicides are increasingly used in controlling oomycete pathogens, such as the late blight agent Phytophthora infestans. In plants, low amounts of Phi induce pathogen resistance through an indirect mode of action. We used iTRAQ-based quantitative proteomics to investigate the effects of phosphite on potato plants before and after infection with P. infestans. Ninety-three (62 up-regulated and 31 down-regulated) differentially regulated proteins, from a total of 1172 reproducibly identified proteins, were identified in the leaf proteome of Phi-treated potato plants. Four days post-inoculation with P. infestans, 16 of the 31 down-regulated proteins remained down-regulated and 42 of the 62 up-regulated proteins remained up-regulated, including 90% of the defense proteins. This group includes pathogenesis-related, stress-responsive, and detoxification-related proteins. Callose deposition and ultrastructural analyses of leaf tissues after infection were used to complement the proteomics approach. This study represents the first comprehensive proteomics analysis of the indirect mode of action of Phi, demonstrating broad effects on plant defense and plant metabolism. The proteomics data and the microscopy study suggest that Phi triggers a hypersensitive response that is responsible for induced resistance of potato leaves against P. infestans. Phosphie triggers complex functional changes in potato leaves that are responsible for the induced resistance against Phytophthora infestans. This article is part of a Special Issue entitled: Translational Plant Proteomics. Copyright © 2013 Elsevier B.V. All rights reserved.

Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome analysis by using a strong cation exchange trap column.

PubMed

Jiang, Xiaogang; Feng, Shun; Tian, Ruijun; Han, Guanghui; Jiang, Xinning; Ye, Mingliang; Zou, Hanfa

2007-02-01

An approach was developed to automate sample introduction for nanoflow LC-MS/MS (microLC-MS/MS) analysis using a strong cation exchange (SCX) trap column. The system consisted of a 100 microm id x 2 cm SCX trap column and a 75 microm id x 12 cm C18 RP analytical column. During the sample loading step, the flow passing through the SCX trap column was directed to waste for loading a large volume of sample at high flow rate. Then the peptides bound on the SCX trap column were eluted onto the RP analytical column by a high salt buffer followed by RP chromatographic separation of the peptides at nanoliter flow rate. It was observed that higher performance of separation could be achieved with the system using SCX trap column than with the system using C18 trap column. The high proteomic coverage using this approach was demonstrated in the analysis of tryptic digest of BSA and yeast cell lysate. In addition, this system was also applied to two-dimensional separation of tryptic digest of human hepatocellular carcinoma cell line SMMC-7721 for large scale proteome analysis. This system was fully automated and required minimum changes on current microLC-MS/MS system. This system represented a promising platform for routine proteome analysis.

Find Pairs: The Module for Protein Quantification of the PeakQuant Software Suite

PubMed Central

Eisenacher, Martin; Kohl, Michael; Wiese, Sebastian; Hebeler, Romano; Meyer, Helmut E.

2012-01-01

Abstract Accurate quantification of proteins is one of the major tasks in current proteomics research. To address this issue, a wide range of stable isotope labeling techniques have been developed, allowing one to quantitatively study thousands of proteins by means of mass spectrometry. In this article, the FindPairs module of the PeakQuant software suite is detailed. It facilitates the automatic determination of protein abundance ratios based on the automated analysis of stable isotope-coded mass spectrometric data. Furthermore, it implements statistical methods to determine outliers due to biological as well as technical variance of proteome data obtained in replicate experiments. This provides an important means to evaluate the significance in obtained protein expression data. For demonstrating the high applicability of FindPairs, we focused on the quantitative analysis of proteome data acquired in 14N/15N labeling experiments. We further provide a comprehensive overview of the features of the FindPairs software, and compare these with existing quantification packages. The software presented here supports a wide range of proteomics applications, allowing one to quantitatively assess data derived from different stable isotope labeling approaches, such as 14N/15N labeling, SILAC, and iTRAQ. The software is publicly available at http://www.medizinisches-proteom-center.de/software and free for academic use. PMID:22909347

Beneficial effects of omega-3 fatty acids in the proteome of high-density lipoprotein proteome

PubMed Central

2012-01-01

Background Omega-3 poly-unsaturated fatty acids (ω-3 PUFAs) have demonstrated to be beneficial in the prevention of cardiovascular disease, however, the mechanisms by which they perform their cardiovascular protection have not been clarified. Intriguingly, some of these protective effects have also been linked to HDL. The hypothesis of this study was that ω-3 PUFAs could modify the protein cargo of HDL particle in a triglyceride non-dependent mode. The objective of the study was to compare the proteome of HDL before and after ω-3 PUFAs supplemented diet. Methods A comparative proteomic analysis in 6 smoker subjects HDL before and after a 5 weeks ω-3 PUFAs enriched diet has been performed. Results Among the altered proteins, clusterin, paraoxonase, and apoAI were found to increase, while fibronectin, α-1-antitrypsin, complement C1r subcomponent and complement factor H decreased after diet supplementation with ω-3 PUFAs. Immunodetection assays confirmed these results. The up-regulated proteins are related to anti-oxidant, anti-inflammatory and anti-atherosclerotic properties of HDL, while the down-regulated proteins are related to regulation of complement activation and acute phase response. Conclusions Despite the low number of subjects included in the study, our findings demonstrate that ω-3 PUFAs supplementation modifies lipoprotein containing apoAI (LpAI) proteome and suggest that these protein changes improve the functionality of the particle. PMID:22978374

A unique proteomic profile on surface IgM ligation in unmutated chronic lymphocytic leukemia

PubMed Central

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

Major depressive disorder: insight into candidate cerebrospinal fluid protein biomarkers from proteomics studies.

PubMed

Al Shweiki, Mhd Rami; Oeckl, Patrick; Steinacker, Petra; Hengerer, Bastian; Schönfeldt-Lecuona, Carlos; Otto, Markus

2017-06-01

Major Depressive Disorder (MDD) is the leading cause of global disability, and an increasing body of literature suggests different cerebrospinal fluid (CSF) proteins as biomarkers of MDD. The aim of this review is to summarize the suggested CSF biomarkers and to analyze the MDD proteomics studies of CSF and brain tissues for promising biomarker candidates. Areas covered: The review includes the human studies found by a PubMed search using the following terms: 'depression cerebrospinal fluid biomarker', 'major depression biomarker CSF', 'depression CSF biomarker', 'proteomics depression', 'proteomics biomarkers in depression', 'proteomics CSF biomarker in depression', and 'major depressive disorder CSF'. The literature analysis highlights promising biomarker candidates and demonstrates conflicting results on others. It reveals 42 differentially regulated proteins in MDD that were identified in more than one proteomics study. It discusses the diagnostic potential of the biomarker candidates and their association with the suggested pathologies. Expert commentary: One ultimate goal of finding biomarkers for MDD is to improve the diagnostic accuracy to achieve better treatment outcomes; due to the heterogeneous nature of MDD, using bio-signatures could be a good strategy to differentiate MDD from other neuropsychiatric disorders. Notably, further validation studies of the suggested biomarkers are still needed.

Assessment of Mitochondrial Dysfunction Arising from Treatment with Hepatotoxicants

PubMed Central

King, Adrienne L.; Bailey, Shannon M.

2010-01-01

Studies demonstrate that mitochondrial dysfunction is a key causative factor in liver disease. Indeed, defects in mitochondrial energy metabolism, disrupted calcium handling, and increased reactive oxygen/nitrogen species production are observed in many metabolic disorders and diseases induced by toxicants. Mitochondria have emerged as a main research focus through work defining new functions of this key organelle in normal cellular physiology and pathophysiology. Specifically, studies show a critical role of mitochondrial reactive oxygen/nitrogen species production in regulating cellular signaling pathways involved in cell survival and death. Given this, along with advances made in proteomics technologies, mitochondria are recognized as top candidates for proteomics analysis. However, assessment of mitochondrial function and it’s proteome following toxicant exposure are not trivial undertakings. In this chapter a technique used to isolate mitochondria from liver tissue is presented along with methods needed to assess mitochondria functionality. The methods described include measurement of mitochondrial respiration, calcium accumulation, and reactive oxygen species production. A presentation of proteomics approaches is also included to allow researchers the basic tools needed to identify alterations in the mitochondrial proteome that contribute to toxicant-mediated diseases. Specifically, methods are presented that demonstrate how thiol labeling reagents in combination with electrophoresis and western blotting can be used to detect oxidant-mediated alterations in mitochondrial protein thiols. A few select pieces data are presented highlighting the power of proteomics to identify mitochondrial targets that contribute to mitochondrial dysfunction and hepatotoxicity in response to specific toxicant exposures and metabolic stressors such as alcohol and environmental tobacco smoke. PMID:23045017

HUPO BPP pilot study: a proteomics analysis of the mouse brain of different developmental stages.

PubMed

Wang, Jing; Gu, Yong; Wang, Lihong; Hang, Xingyi; Gao, Yan; Wang, Hangyan; Zhang, Chenggang

2007-11-01

This study is a part of the HUPO Brain Proteome Project (BPP) pilot study, which aims at obtaining a reliable database of mouse brain proteome, at the comparison of techniques, laboratories, and approaches as well as at preparing subsequent proteome studies of neurologic diseases. The C57/Bl6 mouse brains of three developmental stages at embryonic day 16 (E16), postnatal day 7 (P7), and 8 wk (P56) (n = 5 in each group) were provided by the HUPO BPP executive committee. The whole brain proteins of each animal were individually prepared using 2-DE coupled with PDQuest software analysis. The protein spots representing developmentally related or stably expressed proteins were then prepared with in-gel digestion followed with MALDI-TOF/TOF MS/MS and analyzed using the MASCOT search engines to search the Swiss-Prot or NCBInr database. The 2-DE gel maps of the mouse brains of all of the developmental stages were obtained and submitted to the Data Collection Centre (DCC). The proteins alpha-enolase, stathmin, actin, C14orf166 homolog, 28,000 kDa heat- and acid-stable phosphoprotein, 3-mercaptopyruvate sulfurtransferase and 40 S ribosomal protein S3a were successfully identified. A further Western blotting analysis demonstrated that enolase is a protein up-regulated in the mouse brain from embryonic stage to adult stage. These data are helpful for understanding the proteome changes in the development of the mouse brain.

EBprot: Statistical analysis of labeling-based quantitative proteomics data.

PubMed

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.

Comparative proteomic analysis reveals that T3SS, Tfp, and xanthan gum are key factors in initial stages of Citrus sinensis infection by Xanthomonas citri subsp. citri.

PubMed

Facincani, Agda P; Moreira, Leandro M; Soares, Márcia R; Ferreira, Cristiano B; Ferreira, Rafael M; Ferro, Maria I T; Ferro, Jesus A; Gozzo, Fabio C; de Oliveira, Julio C F

2014-03-01

The bacteria Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus canker. The disease symptoms are characterized by localized host cell hyperplasia followed by tissue necrosis at the infected area. An arsenal of bacterial pathogenicity- and virulence-related proteins is expressed to ensure a successful infection process. At the post-genomic stage of Xac, we used a proteomic approach to analyze the proteins that are displayed differentially over time when the pathogen attacks the host plant. Protein extracts were prepared from infectious Xac grown in inducing medium (XAM1) for 24 h or from host citrus plants for 3 or 5 days after infection, detached times to evaluate the adaptation and virulence of the pathogen. The protein extracts were proteolyzed, and the peptides derived from tryptic digestion were investigated using liquid chromatography and tandem mass spectrometry. Changes in the protein expression profile were compared with the Xac genome and the proteome recently described under non-infectious conditions. An analysis of the proteome of Xac under infectious conditions revealed proteins directly involved in virulence such as the type III secretion system (T3SS) and effector proteins (T3SS-e), the type IV pilus (Tfp), and xanthan gum biosynthesis. Moreover, four new mutants related to proteins detected in the proteome and with different functions exhibited reduced virulence relative to the wild-type proteins. The results of the proteome analysis of infectious Xac define the processes of adaptation to the host and demonstrate the induction of the virulence factors of Xac involved in plant-pathogen interactions.

A Proteomics Approach to Discover Drought Tolerance Proteins in Wheat Pollen Grain at Meiosis Stage.

PubMed

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.

Quantitative HDL Proteomics Identifies Peroxiredoxin-6 as a Biomarker of Human Abdominal Aortic Aneurysm

PubMed Central

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

Protein cleavage strategies for an improved analysis of the membrane proteome

PubMed Central

Fischer, Frank; Poetsch, Ansgar

2006-01-01

Background Membrane proteins still remain elusive in proteomic studies. This is in part due to the distribution of the amino acids lysine and arginine, which are less frequent in integral membrane proteins and almost absent in transmembrane helices. As these amino acids are cleavage targets for the commonly used protease trypsin, alternative cleavage conditions, which should improve membrane protein analysis, were tested by in silico digestion for the three organisms Saccharomyces cerevisiae, Halobacterium sp. NRC-1, and Corynebacterium glutamicum as hallmarks for eukaryotes, archea and eubacteria. Results For the membrane proteomes from all three analyzed organisms, we identified cleavage conditions that achieve better sequence and proteome coverage than trypsin. Greater improvement was obtained for bacteria than for yeast, which was attributed to differences in protein size and GRAVY. It was demonstrated for bacteriorhodopsin that the in silico predictions agree well with the experimental observations. Conclusion For all three examined organisms, it was found that a combination of chymotrypsin and staphylococcal peptidase I gave significantly better results than trypsin. As some of the improved cleavage conditions are not more elaborate than trypsin digestion and have been proven useful in practice, we suppose that the cleavage at both hydrophilic and hydrophobic amino acids should facilitate in general the analysis of membrane proteins for all organisms. PMID:16512920

Application of tissue mesodissection to molecular cancer diagnostics.

PubMed

Krizman, David; Adey, Nils; Parry, Robert

2015-02-01

To demonstrate clinical application of a mesodissection platform that was developed to combine advantages of laser-based instrumentation with the speed/ease of manual dissection for automated dissection of tissue off standard glass slides. Genomic analysis for KRAS gene mutation was performed on formalin fixed paraffin embedded (FFPE) cancer patient tissue that was dissected using the mesodissection platform. Selected reaction monitoring proteomic analysis for quantitative Her2 protein expression was performed on FFPE patient tumour tissue dissected by a laser-based instrument and the MilliSect instrument. Genomic analysis demonstrates highly confident detection of KRAS mutation specifically in lung cancer cells and not the surrounding benign, non-tumour tissue. Proteomic analysis demonstrates Her2 quantitative protein expression in breast cancer cells dissected manually, by laser-based instrumentation and by MilliSect instrumentation (mesodissection). Slide-mounted tissue dissection is commonly performed using laser-based instruments or manually scraping tissue by scalpel. Here we demonstrate that the mesodissection platform as performed by the MilliSect instrument for tissue dissection is cost-effective; it functions comparably to laser-based dissection and which can be adopted into a clinical diagnostic workflow. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Quantitative proteomics reveals a role of JAZ7 in plant defense response to Pseudomonas syringae DC3000.

PubMed

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 experiment suggests that protein abundance is determined by genotype, environment and the interaction between them. Copyright © 2018 Elsevier B.V. All rights reserved.

The iron-responsive microsomal proteome of Aspergillus fumigatus.

PubMed

Moloney, Nicola M; Owens, Rebecca A; Meleady, Paula; Henry, Michael; Dolan, Stephen K; Mulvihill, Eoin; Clynes, Martin; Doyle, Sean

2016-03-16

Aspergillus fumigatus is an opportunistic fungal pathogen. Siderophore biosynthesis and iron acquisition are essential for virulence. Yet, limited data exist with respect to the adaptive nature of the fungal microsomal proteome under iron-limiting growth conditions, as encountered during host infection. Here, we demonstrate that under siderophore biosynthetic conditions--significantly elevated fusarinine C (FSC) and triacetylfusarinine C (TAFC) production (p<0.0001), extensive microsomal proteome remodelling occurs. Specifically, a four-fold enrichment of transmembrane-containing proteins was observed with respect to whole cell lysates following ultracentrifugation-based microsomal extraction. Comparative label-free proteomic analysis of microsomal extracts, isolated following iron-replete and -deplete growth, identified 710 unique proteins. Scatterplot analysis (MaxQuant) demonstrated high correlation amongst biological replicates from each growth condition (Pearson correlation >0.96 within groups; biological replicates (n=4)). Quantitative and qualitative comparison revealed 231 proteins with a significant change in abundance between the iron-replete and iron-deplete conditions (p<0.05, fold change ≥ 2), with 96 proteins showing increased abundance and 135 with decreased abundance following iron limitation, including predicted siderophore transporters. Fluorescently labelled FSC was only sequestered following A. fumigatus growth under iron-limiting conditions. Interestingly, human sera exhibited significantly increased reactivity (p<0.0001) against microsomal protein extracts obtained following iron-deplete growth. The opportunistic fungal pathogen Aspergillus fumigatus must acquire iron to facilitate growth and pathogenicity. Iron-chelating non-ribosomal peptides, termed siderophores, mediate iron uptake via membrane-localised transporter proteins. Here we demonstrate for the first time that growth of A. fumigatus under iron-deplete conditions, concomitant with siderophore biosynthesis, leads to an extensive remodelling of the microsomal proteome which includes significantly altered levels of 231 constituent proteins (96 increased and 135 decreased in abundance), many of which have not previously been localised to the microsome. We also demonstrate the first synthesis of a fluorescent version of fusarinine C, an extracellular A. fumigatus siderophore, and its uptake and localization under iron-restricted conditions. This infers the use of an A. fumigatus siderophore as a 'Trojan horse' to potentiate the efficacy of anti-fungal drugs. Finally, in addition to revealing the Aspergillus-specific IgG reactivity in normal human sera against microsomal proteins, there appears to be a significantly increased reactivity against microsomal proteins obtained following iron-restricted growth. We hypothesise that iron-limiting environment in humans, which has evolved to nutritionally limit pathogen growth in vivo, may also alter the fungal microsomal proteome. Copyright © 2016 Elsevier B.V. All rights reserved.

Label-free Quantitative Proteomics of Mouse Cerebrospinal Fluid Detects β-Site APP Cleaving Enzyme (BACE1) Protease Substrates In Vivo*

PubMed Central

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

Proteome-metabolome profiling of ovarian cancer ascites reveals novel components involved in intercellular communication.

PubMed

Shender, Victoria O; Pavlyukov, Marat S; Ziganshin, Rustam H; Arapidi, Georgij P; Kovalchuk, Sergey I; Anikanov, Nikolay A; Altukhov, Ilya A; Alexeev, Dmitry G; Butenko, Ivan O; Shavarda, Alexey L; Khomyakova, Elena B; Evtushenko, Evgeniy; Ashrafyan, Lev A; Antonova, Irina B; Kuznetcov, Igor N; Gorbachev, Alexey Yu; Shakhparonov, Mikhail I; Govorun, Vadim M

2014-12-01

Ovarian cancer ascites is a native medium for cancer cells that allows investigation of their secretome in a natural environment. This medium is of interest as a promising source of potential biomarkers, and also as a medium for cell-cell communication. The aim of this study was to elucidate specific features of the malignant ascites metabolome and proteome. In order to omit components of the systemic response to ascites formation, we compared malignant ascites with cirrhosis ascites. Metabolome analysis revealed 41 components that differed significantly between malignant and cirrhosis ascites. Most of the identified cancer-specific metabolites are known to be important signaling molecules. Proteomic analysis identified 2096 and 1855 proteins in the ovarian cancer and cirrhosis ascites, respectively; 424 proteins were specific for the malignant ascites. Functional analysis of the proteome demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins. Additionally, we demonstrate that several splicing RNAs were exclusively detected in malignant ascites, where they probably existed within protein complexes. This result was confirmed in vitro using an ovarian cancer cell line. Identification of spliceosomal proteins and RNAs in an extracellular medium is of particular interest; the finding suggests that they might play a role in the communication between cancer cells. In addition, malignant ascites contains a high number of exosomes that are known to play an important role in signal transduction. Thus our study reveals the specific features of malignant ascites that are associated with its function as a medium of intercellular communication. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Experimental Null Method to Guide the Development of Technical Procedures and to Control False-Positive Discovery in Quantitative Proteomics.

PubMed

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-positives/negatives are known and span a wide concentration range. It was observed that the EN method correctly reflects the null distribution in a proteomic system and accurately measures false altered proteins discovery rate (FADR). In summary, the EN method provides a straightforward, practical, and accurate alternative to statistics-based approaches for the development and evaluation of proteomic experiments and can be universally adapted to various types of quantitative techniques.

Time-resolved Analysis of Proteome Dynamics by Tandem Mass Tags and Stable Isotope Labeling in Cell Culture (TMT-SILAC) Hyperplexing*

PubMed Central

Welle, Kevin A.; Zhang, Tian; Hryhorenko, Jennifer R.; Shen, Shichen; Qu, Jun; Ghaemmaghami, Sina

2016-01-01

Recent advances in mass spectrometry have enabled system-wide analyses of protein turnover. By globally quantifying the kinetics of protein clearance and synthesis, these methodologies can provide important insights into the regulation of the proteome under varying cellular and environmental conditions. To facilitate such analyses, we have employed a methodology that combines metabolic isotopic labeling (Stable Isotope Labeling in Cell Culture - SILAC) with isobaric tagging (Tandem Mass Tags - TMT) for analysis of multiplexed samples. The fractional labeling of multiple time-points can be measured in a single mass spectrometry run, providing temporally resolved measurements of protein turnover kinetics. To demonstrate the feasibility of the approach, we simultaneously measured the kinetics of protein clearance and accumulation for more than 3000 proteins in dividing and quiescent human fibroblasts and verified the accuracy of the measurements by comparison to established non-multiplexed approaches. The results indicate that upon reaching quiescence, fibroblasts compensate for lack of cellular growth by globally downregulating protein synthesis and upregulating protein degradation. The described methodology significantly reduces the cost and complexity of temporally-resolved dynamic proteomic experiments and improves the precision of proteome-wide turnover data. PMID:27765818

Determination of burn patient outcome by large-scale quantitative discovery proteomics

PubMed Central

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

Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sivagnanam, Kumaran; Raghavan, Vijaya G. S.; Shah, Manesh B

2011-01-01

Background: Butanol is a second generation biofuel produced by Clostridium acetobutylicum through acetonebutanol- ethanol (ABE) fermentation process. Shotgun proteomics provides a direct approach to study the whole proteome of an organism in depth. This paper focuses on shotgun proteomic profiling of C. acetobutylicum from ABE fermentation using glucose and xylose to understand the functional mechanisms of C. acetobutylicum proteins involved in butanol production. Results: We identified 894 different proteins in C. acetobutylicum from ABE fermentation process by two dimensional - liquid chromatography - tandem mass spectrometry (2D-LC-MS/MS) method. This includes 717 proteins from glucose and 826 proteins from the xylosemore » substrate. A total of 649 proteins were found to be common and 22 significantly differentially expressed proteins were identified between glucose and xylose substrates. Conclusion: Our results demonstrate that flagellar proteins are highly up-regulated with glucose compared to xylose substrate during ABE fermentation. Chemotactic activity was also found to be lost with the xylose substrate due to the absence of CheW and CheV proteins. This is the first report on the shotgun proteomic analysis of C. acetobutylicum ATCC 824 in ABE fermentation between glucose and xylose substrate from a single time data point and the number of proteins identified here is more than any other study performed on this organism up to this report.« less

Abundant storage protein depletion from tuber proteins using ethanol precipitation method: Suitability to proteomics study.

PubMed

Lee, Hye Min; Gupta, Ravi; Kim, Sun Hyung; Wang, Yiming; Rakwal, Randeep; Agrawal, Ganesh Kumar; Kim, Sun Tae

2015-05-01

High-abundance proteins (HAPs) hamper in-depth proteome study necessitating development of a HAPs depletion method. Here, we report a novel ethanol precipitation method (EPM) for HAPs depletion from total tuber proteins. Ethanol showed a dose-dependent effect on depletion of sporamin from sweet potato and patatin from potato tubers, respectively. The 50% ethanol was an optimal concentration. 2DE analysis of EPM-prepared sweet potato proteins also revealed enrichment of storage proteins (SPs) in ethanol supernatant (ES) resulting in detection of new low-abundance proteins in ethanol pellet (EP), compared to total fraction. The ES fraction showed even higher trypsin inhibitor activity than total proteins, further showing the efficacy of EPM in enrichment of sporamin in ES fraction. Application of this method was demonstrated for comparative proteomics of two sweet potato cultivars (Hwang-geum and Ho-bac) and purification of SP (sporamin) in its native form, as examples. Comparative proteomics identified many cultivar specific protein spots and selected spots were confidently assigned for their protein identity using MALDI-TOF-TOF analysis. Overall, the EPM is simple, reproducible, and economical for depletion of SPs and is suitable for downstream proteomics study. This study opens a door for its potential application to other tuber crops or fruits rich in carbohydrates. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mining the human plasma proteome with three-dimensional strategies by high-resolution Quadrupole Orbitrap Mass Spectrometry.

PubMed

Zhao, Yan; Chang, Cheng; Qin, Peibin; Cao, Qichen; Tian, Fang; Jiang, Jing; Li, Xianyu; Yu, Wenfeng; Zhu, Yunping; He, Fuchu; Ying, Wantao; Qian, Xiaohong

2016-01-21

Human plasma is a readily available clinical sample that reflects the status of the body in normal physiological and disease states. Although the wide dynamic range and immense complexity of plasma proteins are obstacles, comprehensive proteomic analysis of human plasma is necessary for biomarker discovery and further verification. Various methods such as immunodepletion, protein equalization and hyper fractionation have been applied to reduce the influence of high-abundance proteins (HAPs) and to reduce the high level of complexity. However, the depth at which the human plasma proteome has been explored in a relatively short time frame has been limited, which impedes the transfer of proteomic techniques to clinical research. Development of an optimal strategy is expected to improve the efficiency of human plasma proteome profiling. Here, five three-dimensional strategies combining HAP depletion (the 1st dimension) and protein fractionation (the 2nd dimension), followed by LC-MS/MS analysis (the 3rd dimension) were developed and compared for human plasma proteome profiling. Pros and cons of the five strategies are discussed for two issues: HAP depletion and complexity reduction. Strategies A and B used proteome equalization and tandem Seppro IgY14 immunodepletion, respectively, as the first dimension. Proteome equalization (strategy A) was biased toward the enrichment of basic and low-molecular weight proteins and had limited ability to enrich low-abundance proteins. By tandem removal of HAPs (strategy B), the efficiency of HAP depletion was significantly increased, whereas more off-target proteins were subtracted simultaneously. In the comparison of complexity reduction, strategy D involved a deglycosylation step before high-pH RPLC separation. However, the increase in sequence coverage did not increase the protein number as expected. Strategy E introduced SDS-PAGE separation of proteins, and the results showed oversampling of HAPs and identification of fewer proteins. Strategy C combined single Seppro IgY14 immunodepletion, high-pH RPLC fractionation and LC-MS/MS analysis. It generated the largest dataset, containing 1544 plasma protein groups and 258 newly identified proteins in a 30-h-machine-time analysis, making it the optimum three-dimensional strategy in our study. Further analysis of the integrated data from the five strategies showed identical distribution patterns in terms of sequence features and GO functional analysis with the 1929-plasma-protein dataset, further supporting the reliability of our plasma protein identifications. The characterization of 20 cytokines in the concentration range from sub-nanograms/milliliter to micrograms/milliliter demonstrated the sensitivity of the current strategies. Copyright © 2015 Elsevier B.V. All rights reserved.

Analysis of Reproducibility of Proteome Coverage and Quantitation Using Isobaric Mass Tags (iTRAQ and TMT).

PubMed

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.

Environmental enrichment alters protein expression as well as the proteomic response to cocaine in rat nucleus accumbens

PubMed Central

Lichti, Cheryl F.; Fan, Xiuzhen; English, Robert D.; Zhang, Yafang; Li, Dingge; Kong, Fanping; Sinha, Mala; Andersen, Clark R.; Spratt, Heidi; Luxon, Bruce A.; Green, Thomas A.

2014-01-01

Prior research demonstrated that environmental enrichment creates individual differences in behavior leading to a protective addiction phenotype in rats. Understanding the mechanisms underlying this phenotype will guide selection of targets for much-needed novel pharmacotherapeutics. The current study investigates differences in proteome expression in the nucleus accumbens of enriched and isolated rats and the proteomic response to cocaine self-administration using a liquid chromatography mass spectrometry (LCMS) technique to quantify 1917 proteins. Results of complementary Ingenuity Pathways Analyses (IPA) and gene set enrichment analyses (GSEA), both performed using protein quantitative data, demonstrate that cocaine increases vesicular transporters for dopamine and glutamate as well as increasing proteins in the RhoA pathway. Further, cocaine regulates proteins related to ERK, CREB and AKT signaling. Environmental enrichment altered expression of a large number of proteins implicated in a diverse number of neuronal functions (e.g., energy production, mRNA splicing, and ubiquitination), molecular cascades (e.g., protein kinases), psychiatric disorders (e.g., mood disorders), and neurodegenerative diseases (e.g., Huntington's and Alzheimer's diseases). Upregulation of energy metabolism components in EC rats was verified using RNA sequencing. Most of the biological functions and pathways listed above were also identified in the Cocaine X Enrichment interaction analysis, providing clear evidence that enriched and isolated rats respond quite differently to cocaine exposure. The overall impression of the current results is that enriched saline-administering rats have a unique proteomic complement compared to enriched cocaine-administering rats as well as saline and cocaine-taking isolated rats. These results identify possible mechanisms of the protective phenotype and provide fertile soil for developing novel pharmacotherapeutics. Proteomics data are available via ProteomeXchange with identifier PXD000990. PMID:25100957

Unintended changes in protein expression revealed by proteomic analysis of seeds from transgenic pea expressing a bean alpha-amylase inhibitor gene.

PubMed

Chen, Hancai; Bodulovic, Greg; Hall, Prudence J; Moore, Andy; Higgins, Thomas J V; Djordjevic, Michael A; Rolfe, Barry G

2009-09-01

Seeds of genetically modified (GM) peas (Pisum sativum L.) expressing the gene for alpha-amylase inhibitor-1 (alphaAI1) from the common bean (Phaseolus vulgaris L. cv. Tendergreen) exhibit resistance to the pea weevil (Bruchus pisorum). A proteomic analysis was carried out to compare seeds from GM pea lines expressing the bean alphaAI1 protein and the corresponding alphaAI1-free segregating lines and non-GM parental line to identify unintended alterations to the proteome of GM peas due to the introduction of the gene for alphaAI1. Proteomic analysis showed that in addition to the presence of alphaAI1, 33 other proteins were differentially accumulated in the alphaAI1-expressing GM lines compared with their non-GM parental line and these were grouped into five expression classes. Among these 33 proteins, only three were found to be associated with the expression of alphaAI1 in the GM pea lines. The accumulation of the remaining 30 proteins appears to be associated with Agrobacterium-mediated transformation events. Sixteen proteins were identified after MALDI-TOF-TOF analysis. About 56% of the identified proteins with altered accumulation in the GM pea were storage proteins including legumin, vicilin or convicilin, phaseolin, cupin and valosin-containing protein. Two proteins were uniquely expressed in the alphaAI1-expressing GM lines and one new protein was present in both the alphaAI1-expressing GM lines and their alphaAI1-free segregating lines, suggesting that both transgenesis and transformation events led to demonstrable changes in the proteomes of the GM lines tested.

Systematic Analysis of Compositional Order of Proteins Reveals New Characteristics of Biological Functions and a Universal Correlate of Macroevolution

PubMed Central

Persi, Erez; Horn, David

2013-01-01

We present a novel analysis of compositional order (CO) based on the occurrence of Frequent amino-acid Triplets (FTs) that appear much more than random in protein sequences. The method captures all types of proteomic compositional order including single amino-acid runs, tandem repeats, periodic structure of motifs and otherwise low complexity amino-acid regions. We introduce new order measures, distinguishing between ‘regularity’, ‘periodicity’ and ‘vocabulary’, to quantify these phenomena and to facilitate the identification of evolutionary effects. Detailed analysis of representative species across the tree-of-life demonstrates that CO proteins exhibit numerous functional enrichments, including a wide repertoire of particular patterns of dependencies on regularity and periodicity. Comparison between human and mouse proteomes further reveals the interplay of CO with evolutionary trends, such as faster substitution rate in mouse leading to decrease of periodicity, while innovation along the human lineage leads to larger regularity. Large-scale analysis of 94 proteomes leads to systematic ordering of all major taxonomic groups according to FT-vocabulary size. This is measured by the count of Different Frequent Triplets (DFT) in proteomes. The latter provides a clear hierarchical delineation of vertebrates, invertebrates, plants, fungi and prokaryotes, with thermophiles showing the lowest level of FT-vocabulary. Among eukaryotes, this ordering correlates with phylogenetic proximity. Interestingly, in all kingdoms CO accumulation in the proteome has universal characteristics. We suggest that CO is a genomic-information correlate of both macroevolution and various protein functions. The results indicate a mechanism of genomic ‘innovation’ at the peptide level, involved in protein elongation, shaped in a universal manner by mutational and selective forces. PMID:24278003

In-depth analysis of the thylakoid membrane proteome of Arabidopsis thaliana chloroplasts: new proteins, new functions, and a plastid proteome database.

PubMed

Friso, Giulia; Giacomelli, Lisa; Ytterberg, A Jimmy; Peltier, Jean-Benoit; Rudella, Andrea; Sun, Qi; Wijk, Klaas J van

2004-02-01

An extensive analysis of the Arabidopsis thaliana peripheral and integral thylakoid membrane proteome was performed by sequential extractions with salt, detergent, and organic solvents, followed by multidimensional protein separation steps (reverse-phase HPLC and one- and two-dimensional electrophoresis gels), different enzymatic and nonenzymatic protein cleavage techniques, mass spectrometry, and bioinformatics. Altogether, 154 proteins were identified, of which 76 (49%) were alpha-helical integral membrane proteins. Twenty-seven new proteins without known function but with predicted chloroplast transit peptides were identified, of which 17 (63%) are integral membrane proteins. These new proteins, likely important in thylakoid biogenesis, include two rubredoxins, a potential metallochaperone, and a new DnaJ-like protein. The data were integrated with our analysis of the lumenal-enriched proteome. We identified 83 out of 100 known proteins of the thylakoid localized photosynthetic apparatus, including several new paralogues and some 20 proteins involved in protein insertion, assembly, folding, or proteolysis. An additional 16 proteins are involved in translation, demonstrating that the thylakoid membrane surface is an important site for protein synthesis. The high coverage of the photosynthetic apparatus and the identification of known hydrophobic proteins with low expression levels, such as cpSecE, Ohp1, and Ohp2, indicate an excellent dynamic resolution of the analysis. The sequential extraction process proved very helpful to validate transmembrane prediction. Our data also were cross-correlated to chloroplast subproteome analyses by other laboratories. All data are deposited in a new curated plastid proteome database (PPDB) with multiple search functions (http://cbsusrv01.tc.cornell.edu/users/ppdb/). This PPDB will serve as an expandable resource for the plant community.

Proteomic profiling of ATM kinase proficient and deficient cell lines upon blockage of proteasome activity☆

PubMed Central

Marzano, Valeria; Santini, Simonetta; Rossi, Claudia; Zucchelli, Mirco; D'Alessandro, Annamaria; Marchetti, Carlo; Mingardi, Michele; Stagni, Venturina; Barilà, Daniela; Urbani, Andrea

2012-01-01

Ataxia Telangiectasia Mutated (ATM) protein kinase is a key effector in the modulation of the functionality of some important stress responses, including DNA damage and oxidative stress response, and its deficiency is the hallmark of Ataxia Telangiectasia (A-T), a rare genetic disorder. ATM modulates the activity of hundreds of target proteins, essential for the correct balance between proliferation and cell death. The aim of this study is to evaluate the phenotypic adaptation at the protein level both in basal condition and in presence of proteasome blockage in order to identify the molecules whose level and stability are modulated through ATM expression. We pursued a comparative analysis of ATM deficient and proficient lymphoblastoid cells by label-free shotgun proteomic experiments comparing the panel of proteins differentially expressed. Through a non-supervised comparative bioinformatic analysis these data provided an insight on the functional role of ATM deficiency in cellular carbohydrate metabolism's regulation. This hypothesis has been demonstrated by targeted metabolic fingerprint analysis SRM (Selected Reaction Monitoring) on specific thermodynamic checkpoints of glycolysis. This article is part of a Special Issue entitled: Translational Proteomics. PMID:22641158

Andromeda: a peptide search engine integrated into the MaxQuant environment.

PubMed

Cox, Jürgen; Neuhauser, Nadin; Michalski, Annette; Scheltema, Richard A; Olsen, Jesper V; Mann, Matthias

2011-04-01

A key step in mass spectrometry (MS)-based proteomics is the identification of peptides in sequence databases by their fragmentation spectra. Here we describe Andromeda, a novel peptide search engine using a probabilistic scoring model. On proteome data, Andromeda performs as well as Mascot, a widely used commercial search engine, as judged by sensitivity and specificity analysis based on target decoy searches. Furthermore, it can handle data with arbitrarily high fragment mass accuracy, is able to assign and score complex patterns of post-translational modifications, such as highly phosphorylated peptides, and accommodates extremely large databases. The algorithms of Andromeda are provided. Andromeda can function independently or as an integrated search engine of the widely used MaxQuant computational proteomics platform and both are freely available at www.maxquant.org. The combination enables analysis of large data sets in a simple analysis workflow on a desktop computer. For searching individual spectra Andromeda is also accessible via a web server. We demonstrate the flexibility of the system by implementing the capability to identify cofragmented peptides, significantly improving the total number of identified peptides.

Nuclear proteome analysis of undifferentiated mouse embryonic stem and germ cells.

PubMed

Buhr, Nicolas; Carapito, Christine; Schaeffer, Christine; Kieffer, Emmanuelle; Van Dorsselaer, Alain; Viville, Stéphane

2008-06-01

Embryonic stem cells (ESCs) and embryonic germ cells (EGCs) provide exciting models for understanding the underlying mechanisms that make a cell pluripotent. Indeed, such understanding would enable dedifferentiation and reprogrammation of any cell type from a patient needing a cell therapy treatment. Proteome analysis has emerged as an important technology for deciphering these biological processes and thereby ESC and EGC proteomes are increasingly studied. Nevertheless, their nuclear proteomes have only been poorly investigated up to now. In order to investigate signaling pathways potentially involved in pluripotency, proteomic analyses have been performed on mouse ESC and EGC nuclear proteins. Nuclei from ESCs and EGCs at undifferentiated stage were purified by subcellular fractionation. After 2-D separation, a subtractive strategy (subtracting culture environment contaminating spots) was applied and a comparison of ESC, (8.5 day post coïtum (dpc))-EGC and (11.5 dpc)-EGC specific nuclear proteomes was performed. A total of 33 ESC, 53 (8.5 dpc)-EGC, and 36 (11.5 dpc)-EGC spots were identified by MALDI-TOF-MS and/or nano-LC-MS/MS. This approach led to the identification of two isoforms (with and without N-terminal acetylation) of a known pluripotency marker, namely developmental pluripotency associated 5 (DPPA5), which has never been identified before in 2-D gel-MS studies of ESCs and EGCs. Furthermore, we demonstrated the efficiency of our subtracting strategy, in association with a nuclear subfractionation by the identification of a new protein (protein arginine N-methyltransferase 7; PRMT7) behaving as proteins involved in pluripotency.

VESPA: software to facilitate genomic annotation of prokaryotic organisms through integration of proteomic and transcriptomic data.

PubMed

Peterson, Elena S; McCue, Lee Ann; Schrimpe-Rutledge, Alexandra C; Jensen, Jeffrey L; Walker, Hyunjoo; Kobold, Markus A; Webb, Samantha R; Payne, Samuel H; Ansong, Charles; Adkins, Joshua N; Cannon, William R; Webb-Robertson, Bobbie-Jo M

2012-04-05

The procedural aspects of genome sequencing and assembly have become relatively inexpensive, yet the full, accurate structural annotation of these genomes remains a challenge. Next-generation sequencing transcriptomics (RNA-Seq), global microarrays, and tandem mass spectrometry (MS/MS)-based proteomics have demonstrated immense value to genome curators as individual sources of information, however, integrating these data types to validate and improve structural annotation remains a major challenge. Current visual and statistical analytic tools are focused on a single data type, or existing software tools are retrofitted to analyze new data forms. We present Visual Exploration and Statistics to Promote Annotation (VESPA) is a new interactive visual analysis software tool focused on assisting scientists with the annotation of prokaryotic genomes though the integration of proteomics and transcriptomics data with current genome location coordinates. VESPA is a desktop Java™ application that integrates high-throughput proteomics data (peptide-centric) and transcriptomics (probe or RNA-Seq) data into a genomic context, all of which can be visualized at three levels of genomic resolution. Data is interrogated via searches linked to the genome visualizations to find regions with high likelihood of mis-annotation. Search results are linked to exports for further validation outside of VESPA or potential coding-regions can be analyzed concurrently with the software through interaction with BLAST. VESPA is demonstrated on two use cases (Yersinia pestis Pestoides F and Synechococcus sp. PCC 7002) to demonstrate the rapid manner in which mis-annotations can be found and explored in VESPA using either proteomics data alone, or in combination with transcriptomic data. VESPA is an interactive visual analytics tool that integrates high-throughput data into a genomic context to facilitate the discovery of structural mis-annotations in prokaryotic genomes. Data is evaluated via visual analysis across multiple levels of genomic resolution, linked searches and interaction with existing bioinformatics tools. We highlight the novel functionality of VESPA and core programming requirements for visualization of these large heterogeneous datasets for a client-side application. The software is freely available at https://www.biopilot.org/docs/Software/Vespa.php.

VESPA: software to facilitate genomic annotation of prokaryotic organisms through integration of proteomic and transcriptomic data

PubMed Central

2012-01-01

Background The procedural aspects of genome sequencing and assembly have become relatively inexpensive, yet the full, accurate structural annotation of these genomes remains a challenge. Next-generation sequencing transcriptomics (RNA-Seq), global microarrays, and tandem mass spectrometry (MS/MS)-based proteomics have demonstrated immense value to genome curators as individual sources of information, however, integrating these data types to validate and improve structural annotation remains a major challenge. Current visual and statistical analytic tools are focused on a single data type, or existing software tools are retrofitted to analyze new data forms. We present Visual Exploration and Statistics to Promote Annotation (VESPA) is a new interactive visual analysis software tool focused on assisting scientists with the annotation of prokaryotic genomes though the integration of proteomics and transcriptomics data with current genome location coordinates. Results VESPA is a desktop Java™ application that integrates high-throughput proteomics data (peptide-centric) and transcriptomics (probe or RNA-Seq) data into a genomic context, all of which can be visualized at three levels of genomic resolution. Data is interrogated via searches linked to the genome visualizations to find regions with high likelihood of mis-annotation. Search results are linked to exports for further validation outside of VESPA or potential coding-regions can be analyzed concurrently with the software through interaction with BLAST. VESPA is demonstrated on two use cases (Yersinia pestis Pestoides F and Synechococcus sp. PCC 7002) to demonstrate the rapid manner in which mis-annotations can be found and explored in VESPA using either proteomics data alone, or in combination with transcriptomic data. Conclusions VESPA is an interactive visual analytics tool that integrates high-throughput data into a genomic context to facilitate the discovery of structural mis-annotations in prokaryotic genomes. Data is evaluated via visual analysis across multiple levels of genomic resolution, linked searches and interaction with existing bioinformatics tools. We highlight the novel functionality of VESPA and core programming requirements for visualization of these large heterogeneous datasets for a client-side application. The software is freely available at https://www.biopilot.org/docs/Software/Vespa.php. PMID:22480257

Laser Capture Microdissection of Pancreatic Acinar Cells to Identify Proteomic Alterations in a Murine Model of Caerulein-Induced Pancreatitis

PubMed Central

Shapiro, John P; Komar, Hannah M; Hancioglu, Baris; Yu, Lianbo; Jin, Ming; Ogata, Yuko; Hart, Phil A; Cruz-Monserrate, Zobeida; Lesinski, Gregory B; Conwell, Darwin L

2017-01-01

Objectives: Chronic pancreatitis (CP) is characterized by inflammation and fibrosis of the pancreas, leading to pain, parenchymal damage, and loss of exocrine and endocrine function. There are currently no curative therapies; diagnosis remains difficult and aspects of pathogenesis remain unclear. Thus, there is a need to identify novel biomarkers to improve diagnosis and understand pathophysiology. We hypothesize that pancreatic acinar regions contain proteomic signatures relevant to disease processes, including secreted proteins that could be detected in biofluids. Methods: Acini from pancreata of mice injected with or without caerulein were collected using laser capture microdissection followed by mass spectrometry analysis. This protocol enabled high-throughput analysis that captured altered protein expression throughout the stages of CP. Results: Over 2,900 proteins were identified, whereas 331 were significantly changed ≥2-fold by mass spectrometry spectral count analysis. Consistent with pathogenesis, we observed increases in proteins related to fibrosis (e.g., collagen, P<0.001), several proteases (e.g., trypsin 1, P<0.001), and altered expression of proteins associated with diminished pancreas function (e.g., lipase, amylase, P<0.05). In comparison with proteomic data from a public data set of CP patients, a significant correlation was observed between proteomic changes in tissue from both the caerulein model and CP patients (r=0.725, P<0.001). CONCLUSIONS: This study illustrates the ability to characterize proteome changes of acinar cells isolated from pancreata of caerulein-treated mice and demonstrates a relationship between signatures from murine and human CP. PMID:28406494

Application of targeted quantitative proteomics analysis in human cerebrospinal fluid using a liquid chromatography matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (LC MALDI TOF/TOF) platform.

PubMed

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.

TRIC: an automated alignment strategy for reproducible protein quantification in targeted proteomics

PubMed Central

Röst, Hannes L.; Liu, Yansheng; D’Agostino, Giuseppe; Zanella, Matteo; Navarro, Pedro; Rosenberger, George; Collins, Ben C.; Gillet, Ludovic; Testa, Giuseppe; Malmström, Lars; Aebersold, Ruedi

2016-01-01

Large scale, quantitative proteomic studies have become essential for the analysis of clinical cohorts, large perturbation experiments and systems biology studies. While next-generation mass spectrometric techniques such as SWATH-MS have substantially increased throughput and reproducibility, ensuring consistent quantification of thousands of peptide analytes across multiple LC-MS/MS runs remains a challenging and laborious manual process. To produce highly consistent and quantitatively accurate proteomics data matrices in an automated fashion, we have developed the TRIC software which utilizes fragment ion data to perform cross-run alignment, consistent peak-picking and quantification for high throughput targeted proteomics. TRIC uses a graph-based alignment strategy based on non-linear retention time correction to integrate peak elution information from all LC-MS/MS runs acquired in a study. When compared to state-of-the-art SWATH-MS data analysis, the algorithm was able to reduce the identification error by more than 3-fold at constant recall, while correcting for highly non-linear chromatographic effects. On a pulsed-SILAC experiment performed on human induced pluripotent stem (iPS) cells, TRIC was able to automatically align and quantify thousands of light and heavy isotopic peak groups and substantially increased the quantitative completeness and biological information in the data, providing insights into protein dynamics of iPS cells. Overall, this study demonstrates the importance of consistent quantification in highly challenging experimental setups, and proposes an algorithm to automate this task, constituting the last missing piece in a pipeline for automated analysis of massively parallel targeted proteomics datasets. PMID:27479329

Nanodroplet processing platform for deep and quantitative proteome profiling of 10-100 mammalian cells.

PubMed

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.

Role of the visual experience-dependent nascent proteome in neuronal plasticity

PubMed Central

Liu, Han-Hsuan; McClatchy, Daniel B; Schiapparelli, Lucio; Shen, Wanhua; Yates, John R

2018-01-01

Experience-dependent synaptic plasticity refines brain circuits during development. To identify novel protein synthesis-dependent mechanisms contributing to experience-dependent plasticity, we conducted a quantitative proteomic screen of the nascent proteome in response to visual experience in Xenopus optic tectum using bio-orthogonal metabolic labeling (BONCAT). We identified 83 differentially synthesized candidate plasticity proteins (CPPs). The CPPs form strongly interconnected networks and are annotated to a variety of biological functions, including RNA splicing, protein translation, and chromatin remodeling. Functional analysis of select CPPs revealed the requirement for eukaryotic initiation factor three subunit A (eIF3A), fused in sarcoma (FUS), and ribosomal protein s17 (RPS17) in experience-dependent structural plasticity in tectal neurons and behavioral plasticity in tadpoles. These results demonstrate that the nascent proteome is dynamic in response to visual experience and that de novo synthesis of machinery that regulates RNA splicing and protein translation is required for experience-dependent plasticity. PMID:29412139

Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells

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

Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells

DOE PAGES

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

Proteomic analysis of Chlorella vulgaris: Potential targets for enhanced lipid accumulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guarnieri, Michael T.; Nag, Ambarish; Yang, Shihui

2013-11-01

Oleaginous microalgae are capable of producing large quantities of fatty acids and triacylglycerides. As such, they are promising feedstocks for the production of biofuels and bioproducts. Genetic strain-engineering strategies offer a means to accelerate the commercialization of algal biofuels by improving the rate and total accumulation of microalgal lipids. However, the industrial potential of these organisms remains to be met, largely due to the incomplete knowledgebase surrounding the mechanisms governing the induction of algal lipid biosynthesis. Such strategies require further elucidation of genes and gene products controlling algal lipid accumulation. In this study, we have set out to examine thesemore » mechanisms and identify novel strain-engineering targets in the oleaginous microalga, Chlorella vulgaris. Comparative shotgun proteomic analyses have identified a number of novel targets, including previously unidentified transcription factors and proteins involved in cell signaling and cell cycle regulation. These results lay the foundation for strain-improvement strategies and demonstrate the power of translational proteomic analysis.« less

Elucidating Proteoform Families from Proteoform Intact-Mass and Lysine-Count Measurements

PubMed Central

2016-01-01

Proteomics is presently dominated by the “bottom-up” strategy, in which proteins are enzymatically digested into peptides for mass spectrometric identification. Although this approach is highly effective at identifying large numbers of proteins present in complex samples, the digestion into peptides renders it impossible to identify the proteoforms from which they were derived. We present here a powerful new strategy for the identification of proteoforms and the elucidation of proteoform families (groups of related proteoforms) from the experimental determination of the accurate proteoform mass and number of lysine residues contained. Accurate proteoform masses are determined by standard LC–MS analysis of undigested protein mixtures in an Orbitrap mass spectrometer, and the lysine count is determined using the NeuCode isotopic tagging method. We demonstrate the approach in analysis of the yeast proteome, revealing 8637 unique proteoforms and 1178 proteoform families. The elucidation of proteoforms and proteoform families afforded here provides an unprecedented new perspective upon proteome complexity and dynamics. PMID:26941048

Novel Phage Group Infecting Lactobacillus delbrueckii subsp. lactis, as Revealed by Genomic and Proteomic Analysis of Bacteriophage Ldl1

PubMed Central

Casey, Eoghan; Mahony, Jennifer; Neve, Horst; Noben, Jean-Paul; Dal Bello, Fabio

2014-01-01

Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 ± 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species. PMID:25501478

Targeted proteomic assays for quantitation of proteins identified by proteogenomic analysis of ovarian cancer

DOE PAGES

Song, Ehwang; Gao, Yuqian; Wu, Chaochao; ...

2017-07-19

Here, mass spectrometry (MS) based targeted proteomic methods such as selected reaction monitoring (SRM) are becoming the method of choice for preclinical verification of candidate protein biomarkers. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute has investigated the standardization and analytical validation of the SRM assays and demonstrated robust analytical performance on different instruments across different laboratories. An Assay Portal has also been established by CPTAC to provide the research community a resource consisting of large set of targeted MS-based assays, and a depository to share assays publicly, providing that assays meet the guidelines proposed bymore » CPTAC. Herein, we report 98 SRM assays covering 70 candidate protein biomarkers previously reported as associated with ovarian cancer that have been thoroughly characterized according to the CPTAC Assay Characterization Guidance Document. The experiments, methods and results for characterizing these SRM assays for their MS response, repeatability, selectivity, stability, and reproducible detection of endogenous analytes are described in detail.« less

A novel strategy for global analysis of the dynamic thiol redox proteome.

PubMed

Martínez-Acedo, Pablo; Núñez, Estefanía; Gómez, Francisco J Sánchez; Moreno, Margoth; Ramos, Elena; Izquierdo-Álvarez, Alicia; Miró-Casas, Elisabet; Mesa, Raquel; Rodriguez, Patricia; Martínez-Ruiz, Antonio; Dorado, David Garcia; Lamas, Santiago; Vázquez, Jesús

2012-09-01

Nitroxidative stress in cells occurs mainly through the action of reactive nitrogen and oxygen species (RNOS) on protein thiol groups. Reactive nitrogen and oxygen species-mediated protein modifications are associated with pathophysiological states, but can also convey physiological signals. Identification of Cys residues that are modified by oxidative stimuli still poses technical challenges and these changes have never been statistically analyzed from a proteome-wide perspective. Here we show that GELSILOX, a method that combines a robust proteomics protocol with a new computational approach that analyzes variance at the peptide level, allows a simultaneous analysis of dynamic alterations in the redox state of Cys sites and of protein abundance. GELSILOX permits the characterization of the major endothelial redox targets of hydrogen peroxide in endothelial cells and reveals that hypoxia induces a significant increase in the status of oxidized thiols. GELSILOX also detected thiols that are redox-modified by ischemia-reperfusion in heart mitochondria and demonstrated that these alterations are abolished in ischemia-preconditioned animals.

Characterization of Native Protein Complexes and Protein Isoform Variation Using Size-fractionation-based Quantitative Proteomics*

PubMed Central

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

Characterization of native protein complexes and protein isoform variation using size-fractionation-based quantitative proteomics.

PubMed

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.

Proteomic analysis of human dental cementum and alveolar bone

PubMed Central

Salmon, Cristiane R.; Tomazela, Daniela M.; Ruiz, Karina Gonzales Silvério; Foster, Brian L.; Leme, Adriana Franco Paes; Sallum, Enilson Antonio; Somerman, Martha J.; Nociti, Francisco H.

2013-01-01

Dental cementum (DC) is a bone-like tissue covering the tooth root and responsible for attaching the tooth to the alveolar bone (AB) via the periodontal ligament (PDL). Studies have unsuccessfully tried to identify factors specific to DC versus AB, in an effort to better understand DC development and regeneration. The present study aimed to use matched human DC and AB samples (n=7) to generate their proteomes for comparative analysis. Bone samples were harvested from tooth extraction sites, whereas DC samples were obtained from the apical root portion of extracted third molars. Samples were denatured, followed by protein extraction reduction, alkylation and digestion for analysis by nanoAcquity HPLC system and LTQ-FT Ultra. Data analysis demonstrated that a total of 318 proteins were identified in AB and DC. In addition to shared proteins between these tissues, 105 and 83 proteins exclusive to AB or DC were identified, respectively. This is the first report analyzing the proteomic composition of human DC matrix and identifying putative unique and enriched proteins in comparison to alveolar bone. These findings may provide novel insights into developmental differences between DC and AB, and identify candidate biomarkers that may lead to more efficient and predictable therapies for periodontal regeneration. PMID:24007660

GProX, a user-friendly platform for bioinformatics analysis and visualization of quantitative proteomics data.

PubMed

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.

GProX, a User-Friendly Platform for Bioinformatics Analysis and Visualization of Quantitative Proteomics Data*

PubMed Central

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

Comparative proteomic analysis reveals alterations in development and photosynthesis-related proteins in diploid and triploid rice.

PubMed

Wang, Shuzhen; Chen, Wenyue; Yang, Changdeng; Yao, Jian; Xiao, Wenfei; Xin, Ya; Qiu, Jieren; Hu, Weimin; Yao, Haigen; Ying, Wu; Fu, Yaping; Tong, Jianxin; Chen, Zhongzhong; Ruan, Songlin; Ma, Huasheng

2016-09-13

Polyploidy has pivotal influences on rice (Oryza sativa L.) morphology and physiology, and is very important for understanding rice domestication and improving agricultural traits. Diploid (DP) and triploid (TP) rice shows differences in morphological parameters, such as plant height, leaf length, leaf width and the physiological index of chlorophyll content. However, the underlying mechanisms determining these morphological differences are remain to be defined. To better understand the proteomic changes between DP and TP, tandem mass tags (TMT) mass spectrometry (MS)/MS was used to detect the significant changes to protein expression between DP and TP. Results indicated that both photosynthesis and metabolic pathways were highly significantly associated with proteomic alteration between DP and TP based on biological process and pathway enrichment analysis, and 13 higher abundance chloroplast proteins involving in these two pathways were identified in TP. Quantitative real-time PCR analysis demonstrated that 5 of the 13 chloroplast proteins ATPF, PSAA, PSAB, PSBB and RBL in TP were higher abundance compared with those in DP. This study integrates morphology, physiology and proteomic profiling alteration of DP and TP to address their underlying different molecular mechanisms. Our finding revealed that ATPF, PSAA, PSAB, PSBB and RBL can induce considerable expression changes in TP and may affect the development and growth of rice through photosynthesis and metabolic pathways.

An Integrated Proteomics and Bioinformatics Approach Reveals the Anti-inflammatory Mechanism of Carnosic Acid

PubMed Central

Wang, Li-Chao; Wei, Wen-Hui; Zhang, Xiao-Wen; Liu, Dan; Zeng, Ke-Wu; Tu, Peng-Fei

2018-01-01

Drastic macrophages activation triggered by exogenous infection or endogenous stresses is thought to be implicated in the pathogenesis of various inflammatory diseases. Carnosic acid (CA), a natural phenolic diterpene extracted from Salvia officinalis plant, has been reported to possess anti-inflammatory activity. However, its role in macrophages activation as well as potential molecular mechanism is largely unexplored. In the current study, we sought to elucidate the anti-inflammatory property of CA using an integrated approach based on unbiased proteomics and bioinformatics analysis. CA significantly inhibited the robust increase of nitric oxide and TNF-α, downregulated COX2 protein expression, and lowered the transcriptional level of inflammatory genes including Nos2, Tnfα, Cox2, and Mcp1 in LPS-stimulated RAW264.7 cells, a murine model of peritoneal macrophage cell line. The LC-MS/MS-based shotgun proteomics analysis showed CA negatively regulated 217 LPS-elicited proteins which were involved in multiple inflammatory processes including MAPK, nuclear factor (NF)-κB, and FoxO signaling pathways. A further molecular biology analysis revealed that CA effectually inactivated IKKβ/IκB-α/NF-κB, ERK/JNK/p38 MAPKs, and FoxO1/3 signaling pathways. Collectively, our findings demonstrated the role of CA in regulating inflammation response and provide some insights into the proteomics-guided pharmacological mechanism study of natural products. PMID:29713284

Plasma proteomic changes during hypothermic and normothermic cardiopulmonary bypass in aortic surgeries

PubMed Central

ODA, TEIJI; YAMAGUCHI, AKANE; YOKOYAMA, MASAO; SHIMIZU, KOJI; TOYOTA, KOSAKU; NIKAI, TETSURO; MATSUMOTO, KEN-ICHI

2014-01-01

Deep hypothermic circulatory arrest (DHCA) is a protective method against brain ischemia in aortic surgery. However, the possible effects of DHCA on the plasma proteins remain to be determined. In the present study, we used novel high-throughput technology to compare the plasma proteomes during DHCA (22°C) with selective cerebral perfusion (SCP, n=7) to those during normothermic cardiopulmonary bypass (CPB, n=7). Three plasma samples per patient were obtained during CPB: T1, prior to cooling; T2, during hypothermia; T3, after rewarming for the DHCA group and three corresponding points for the normothermic group. A proteomic analysis was performed using isobaric tag for relative and absolute quantification (iTRAQ) labeling tandem mass spectrometry to assess quantitative protein changes. In total, the analysis identified 262 proteins. The bioinformatics analysis revealed a significant upregulation of complement activation at T2 in normothermic CPB, which was suppressed in DHCA. These findings were confirmed by the changes of the terminal complement complex (SC5b-9) levels. At T3, however, the level of SC5b-9 showed a greater increase in DHCA compared to normothermic CPB, while 48 proteins were significantly downregulated in DHCA. The results demonstrated that DHCA and rewarming potentially exert a significant effect on the plasma proteome in patients undergoing aortic surgery. PMID:25050567

iTRAQ-Based Quantitative Proteomic Analysis of the Initiation of Head Regeneration in Planarians.

PubMed

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.

Notice of Pre-Application Webinar (RFA-CA-15-021, RFA-CA-15-022, RFA-CA-15-023) | Office of Cancer Clinical Proteomics Research

Cancer.gov

The National Cancer Institute will hold a public pre-application webinar on Friday, December 11 at 12:00 p.m. (EST) for the Funding Opportunity Announcements (FOAs) RFA-CA-15-021 entitled “Proteome Characterization Centers for Clinical Proteomic Tumor Analysis Consortium (U24), RFA-CA-15-022 entitled “Proteogenomic Translational Research Centers for Clinical Proteomic Tumor Analysis Consortium (U01)”, and RFA-CA-15-023 entitled “Proteogenomic Data Analysis Centers for Clinical Proteomic Tumor Analysis Consortium (U24)”.

Ultrahigh pressure fast size exclusion chromatography for top-down proteomics.

PubMed

Chen, Xin; Ge, Ying

2013-09-01

Top-down MS-based proteomics has gained a solid growth over the past few years but still faces significant challenges in the LC separation of intact proteins. In top-down proteomics, it is essential to separate the high mass proteins from the low mass species due to the exponential decay in S/N as a function of increasing molecular mass. SEC is a favored LC method for size-based separation of proteins but suffers from notoriously low resolution and detrimental dilution. Herein, we reported the use of ultrahigh pressure (UHP) SEC for rapid and high-resolution separation of intact proteins for top-down proteomics. Fast separation of intact proteins (6-669 kDa) was achieved in < 7 min with high resolution and high efficiency. More importantly, we have shown that this UHP-SEC provides high-resolution separation of intact proteins using a MS-friendly volatile solvent system, allowing the direct top-down MS analysis of SEC-eluted proteins without an additional desalting step. Taken together, we have demonstrated that UHP-SEC is an attractive LC strategy for the size separation of proteins with great potential for top-down proteomics. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The temporal analysis of yeast exponential phase using shotgun proteomics as a fermentation monitoring technique

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Eric L.; Orsat, Valerie; Shah, Manesh B

2012-01-01

System biology and bioprocess technology can be better understood using shotgun proteomics as a monitoring system during the fermentation. We demonstrated a shotgun proteomic method to monitor the temporal yeast proteome in early, middle and late exponential phases. Our study identified a total of 1389 proteins combining all 2D-LC-MS/MS runs. The temporal Saccharomyces cerevisiae proteome was enriched with proteolysis, radical detoxification, translation, one-carbon metabolism, glycolysis and TCA cycle. Heat shock proteins and proteins associated with oxidative stress response were found throughout the exponential phase. The most abundant proteins observed were translation elongation factors, ribosomal proteins, chaperones and glycolytic enzymes. Themore » high abundance of the H-protein of the glycine decarboxylase complex (Gcv3p) indicated the availability of glycine in the environment. We observed differentially expressed proteins and the induced proteins at mid-exponential phase were involved in ribosome biogenesis, mitochondria DNA binding/replication and transcriptional activator. Induction of tryptophan synthase (Trp5p) indicated the abundance of tryptophan during the fermentation. As fermentation progressed toward late exponential phase, a decrease in cell proliferation was implied from the repression of ribosomal proteins, transcription coactivators, methionine aminopeptidase and translation-associated proteins.« less

Unraveling the resistance of microbial biofilms: has proteomics been helpful?

PubMed

Seneviratne, C Jayampath; Wang, Yu; Jin, Lijian; Wong, Sarah S W; Herath, Thanuja D K; Samaranayake, Lakshman P

2012-02-01

Biofilms are surface-attached, matrix-encased, structured microbial communities which display phenotypic features that are dramatically different from those of their free-floating, or planktonic, counterparts. Biofilms seem to be the preferred mode of growth of microorganisms in nature, and at least 65% of all human infections are associated with biofilms. The most notable and clinically relevant property of biofilms is their greater resistance to antimicrobials compared with their planktonic counterparts. Although both bacterial and fungal biofilms display this phenotypic feature, the exact mechanisms underlying their increased drug resistance are yet to be determined. Advances in proteomics techniques during the past decade have facilitated in-depth analysis of the possible mechanisms underpinning increased drug resistance in biofilms. These studies have demonstrated the ability of proteomics techniques to unravel new targets for combating microbial biofilms. In this review, we discuss the putative drug resistance mechanisms of microbial biofilms that have been uncovered by proteomics and critically evaluate the possible contribution of the new knowledge to future development in the field. We also summarize strategic uses of novel proteomics technologies in studies related to drug resistance mechanisms of microbial biofilms. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Insight from Mitochondrial Functions and Proteomics to Understand Cardiometabolic Disorders in Survivors of Acute Lymphoblastic Leukemia.

PubMed

Leahy, Jade; Spahis, Schohraya; Bonneil, Eric; Garofalo, Carole; Grimard, Guy; Morel, Sophia; Laverdière, Caroline; Krajinovic, Maja; Drouin, Simon; Delvin, Edgard; Sinnett, Daniel; Marcil, Valérie; Levy, Emile

2018-03-18

Childhood acute lymphoblastic leukemia (cALL) is the most prevalent form of cancer in children. Due to advances in treatment and therapy, young cALL subjects now achieve a 90% survival rate. However, this tremendous advance does not come without consequence since ~2/3 of cALL survivors are affected by long-term and late, severe complications. Although the metabolic syndrome is a very serious sequel of cALL, the mechanisms remain undefined. It is also surprising to note that the mitochondrion, a central organelle in metabolic functions and the main cellular energy generator, have not yet been explored. To determine whether cALL survivors exhibit impairments in their mitochondrial functions and proteomic profiling in relationship with metabolic disorders in cALL survivors compared to healthy controls. Anthropometric measures, metabolic characteristics and lipid profiles were assessed, mitochondria isolated from peripheral blood mononuclear cells, and proteomic analyzed. Our data demonstrated that metabolically Unhealthy survivors exhibited several metabolic syndrome components (e.g. overweight, insulin resistance, dyslipidemia, inflammation) whereas Healthy cALL survivors resemble the Controls. In line with these abnormalities, functional experiments in these subjects revealed a significant decrease in the protein expression of mitochondrial antioxidant superoxide dismutase, PGC1-α transcription factor (a key modulator of mitochondrion biogenesis), and an increase in pro-apoptotic cytochrome c. Proteomic analysis of mitochondria by mass spectrometry revealed changes in the regulation of proteins related to inflammation, apoptosis, energy production, redox and antioxidant activity, fatty acid β-oxidation, protein transport and metabolism, and signalling pathways between groups. Through the use of proteomic analysis, our work demonstrated a number of significant alterations in protein expression in mitochondria of cALL survivors, especially the metabolically Unhealthy survivor group. Further investigation of these proteins may help delineate the mechanisms by which mitochondrial dysfunctions exert cardiometabolic derangements in cALL survivors. Copyright © 2018. Published by Elsevier Inc.

Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database.

PubMed

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 genome sequences, though there are over lapped proteins. Based on the demonstrated application of data stored in the database for functional analyses, it is suggested that these data will be useful for analyses of biological mechanisms in soybean. Furthermore, coupled with recent advances in information and communication technology, the usefulness of this database would increase in the analyses of biological mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative trait loci mapping of the mouse plasma proteome (pQTL).

PubMed

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.

Quantitative Trait Loci Mapping of the Mouse Plasma Proteome (pQTL)

PubMed Central

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

PeptideDepot: flexible relational database for visual analysis of quantitative proteomic data and integration of existing protein information.

PubMed

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.

Proteome-Wide Profiling of Targets of Cysteine reactive Small Molecules by Using Ethynyl Benziodoxolone Reagents.

PubMed

Abegg, Daniel; Frei, Reto; Cerato, Luca; Prasad Hari, Durga; Wang, Chao; Waser, Jerome; Adibekian, Alexander

2015-09-07

In this study, we present a highly efficient method for proteomic profiling of cysteine residues in complex proteomes and in living cells. Our method is based on alkynylation of cysteines in complex proteomes using a "clickable" alkynyl benziodoxolone bearing an azide group. This reaction proceeds fast, under mild physiological conditions, and with a very high degree of chemoselectivity. The formed azide-capped alkynyl-cysteine adducts are readily detectable by LC-MS/MS, and can be further functionalized with TAMRA or biotin alkyne via CuAAC. We demonstrate the utility of alkynyl benziodoxolones for chemical proteomics applications by identifying the proteomic targets of curcumin, a diarylheptanoid natural product that was and still is part of multiple human clinical trials as anticancer agent. Our results demonstrate that curcumin covalently modifies several key players of cellular signaling and metabolism, most notably the enzyme casein kinase I gamma. We anticipate that this new method for cysteine profiling will find broad application in chemical proteomics and drug discovery. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fungal proteomics: from identification to function.

PubMed

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.

Quantitative proteomic analysis of microdissected oral epithelium for cancer biomarker discovery.

PubMed

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.

Analysis of the liver mitochondrial proteome in response to ethanol and S-adenosylmethionine treatments: novel molecular targets of disease and hepatoprotection.

PubMed

Andringa, Kelly K; King, Adrienne L; Eccleston, Heather B; Mantena, Sudheer K; Landar, Aimee; Jhala, Nirag C; Dickinson, Dale A; Squadrito, Giuseppe L; Bailey, Shannon M

2010-05-01

S-adenosylmethionine (SAM) minimizes alcohol hepatotoxicity; however, the molecular mechanisms responsible for SAM hepatoprotection remain unknown. Herein, we use proteomics to determine whether the hepatoprotective action of SAM against early-stage alcoholic liver disease is linked to alterations in the mitochondrial proteome. For this, male rats were fed control or ethanol-containing liquid diets +/- SAM and liver mitochondria were prepared for proteomic analysis. Two-dimensional isoelectric focusing (2D IEF/SDS-PAGE) and blue native gel electrophoresis (BN-PAGE) were used to determine changes in matrix and oxidative phosphorylation (OxPhos) proteins, respectively. SAM coadministration minimized alcohol-dependent inflammation and preserved mitochondrial respiration. SAM supplementation preserved liver SAM levels in ethanol-fed rats; however, mitochondrial SAM levels were increased by ethanol and SAM treatments. With use of 2D IEF/SDS-PAGE, 30 proteins showed significant changes in abundance in response to ethanol, SAM, or both. Classes of proteins affected by ethanol and SAM treatments were chaperones, beta oxidation proteins, sulfur metabolism proteins, and dehydrogenase enzymes involved in methionine, glycine, and choline metabolism. BN-PAGE revealed novel changes in the levels of 19 OxPhos proteins in response to ethanol, SAM, or both. Ethanol- and SAM-dependent alterations in the proteome were not linked to corresponding changes in gene expression. In conclusion, ethanol and SAM treatment led to multiple changes in the liver mitochondrial proteome. The protective effects of SAM against alcohol toxicity are mediated, in part, through maintenance of proteins involved in key mitochondrial energy conserving and biosynthetic pathways. This study demonstrates that SAM may be a promising candidate for treatment of alcoholic liver disease.

The Skeleton Forming Proteome of an Early Branching Metazoan: A Molecular Survey of the Biomineralization Components Employed by the Coralline Sponge Vaceletia Sp.

PubMed Central

Wörheide, Gert; Jackson, Daniel John

2015-01-01

The ability to construct a mineralized skeleton was a major innovation for the Metazoa during their evolution in the late Precambrian/early Cambrian. Porifera (sponges) hold an informative position for efforts aimed at unraveling the origins of this ability because they are widely regarded to be the earliest branching metazoans, and are among the first multi-cellular animals to display the ability to biomineralize in the fossil record. Very few biomineralization associated proteins have been identified in sponges so far, with no transcriptome or proteome scale surveys yet available. In order to understand what genetic repertoire may have been present in the last common ancestor of the Metazoa (LCAM), and that may have contributed to the evolution of the ability to biocalcify, we have studied the skeletal proteome of the coralline demosponge Vaceletia sp. and compare this to other metazoan biomineralizing proteomes. We bring some spatial resolution to this analysis by dividing Vaceletia’s aragonitic calcium carbonate skeleton into “head” and “stalk” regions. With our approach we were able to identify 40 proteins from both the head and stalk regions, with many of these sharing some similarity to previously identified gene products from other organisms. Among these proteins are known biomineralization compounds, such as carbonic anhydrase, spherulin, extracellular matrix proteins and very acidic proteins. This report provides the first proteome scale analysis of a calcified poriferan skeletal proteome, and its composition clearly demonstrates that the LCAM contributed several key enzymes and matrix proteins to its descendants that supported the metazoan ability to biocalcify. However, lineage specific evolution is also likely to have contributed significantly to the ability of disparate metazoan lineages to biocalcify. PMID:26536128

Identification and Characterization of Soluble Factors Involved in Delayed Effects of Low Dose Radiation. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baulch, Janet

2013-09-11

This is a 'glue grant' that was part of a DOE Low Dose project entitled 'Identification and Characterization of Soluble Factors Involved in Delayed Effects of Low Dose Radiation'. This collaborative program has involved Drs. David L. Springer from Pacific Northwest National Laboratory (PNNL), John H. Miller from Washington State University, Tri-cities (WSU) and William F. Morgan then from the University of Maryland, Baltimore (UMB). In July 2008, Dr. Morgan moved to PNNL and Dr. Janet E. Baulch became PI for this project at University of Maryland. In November of 2008, a one year extension with no new funds wasmore » requested to complete the proteomic analyses. The project stemmed from studies in the Morgan laboratory demonstrating that genomically unstable cells secret a soluble factor or factors into the culture medium, that cause cytogenetic aberrations and apoptosis in normal parental GM10115 cells. The purpose of this project was to identify the death inducing effect (DIE) factor or factors, estimate their relative abundance, identify the cell signaling pathways involved and finally recapitulate DIE in normal cells by exogenous manipulation of putative DIE factors in culture medium. As reported in detail in the previous progress report, analysis of culture medium from the parental cell line, and stable and unstable clones demonstrated inconsistent proteomic profiles as relate to candidate DIE factors. While the proposed proteomic analyses did not provide information that would allow DIE factors to be identified, the analyses provided another important set of observations. Proteomic analysis suggested that proteins associated with the cellular response to oxidative stress and mitochondrial function were elevated in the medium from unstable clones in a manner consistent with mitochondrial dysfunction. These findings correlate with previous studies of these clones that demonstrated functional differences between the mitochondria of stable and unstable clones. These mitochondrial abnormalities in the unstable clones contributes to oxidative stress.« less

Perturbations in the Urinary Exosome in Transplant Rejection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sigdel, Tara K.; NG, Yolanda; Lee, Sangho

Background: Urine exosomes, vesicles exocytosed into urine by all renal epithelial cell types, occur under normal physiologic and disease states. Exosome contents may mirror disease-specific proteome perturbations in kidney injury. Analysis methodologies for the exosomal fraction of the urinary proteome were developed and for comparing the urinary exosomal fraction versus unfractionated proteome for biomarker discovery. Methods: Urine exosomes were isolated by centrifugal filtration from mid-stream, second morning void, urine samples collected from kidney transplant recipients with and without biopsy matched acute rejection. The proteomes of unfractionated whole urine (Uw) and urine exosomes (Uexo) underwent mass spectrometry-based quantitative proteomics analysis. Themore » proteome data were analyzed for significant differential protein abundances in acute rejection (AR). Results: Identifications of 1018 and 349 proteins, Uw and Uexo fractions, respectively, demonstrated a 279 protein overlap between the two urinary compartments with 25%(70) of overlapping proteins unique to Uexoand represented membrane bound proteins (p=9.31e-7). Of 349 urine exosomal proteins identified in transplant patients 220 were not previously identified in the normal urine exosomal fraction. Uexo proteins (11), functioning in the inflammatory / stress response, were more abundant in patients with biopsy-confirmed acute rejection, 3 of which were exclusive to Uexo. Uexo AR-specific biomarkers (8) were also detected in Uw, but since they were observed at significantly lower abundances in Uw, they were not significant for AR in Uw. Conclusions: A rapid urinary exosome isolation method and quantitative measurement of enriched Uexo proteins was applied. Urine proteins specific to the exosomal fraction were detected either in unfractionated urine (at low abundances) or by Uexo fraction analysis. Perturbed proteins in the exosomal compartment of urine collected from kidney transplant patients were specific to inflammatory responses, and were not observed in the Uexo fraction from normal healthy subjects. Uexo specific protein alterations in renal disease provide potential mechanistic insights and offer a unique panel of sensitive biomarkers for monitoring for acute transplant rejection.« less

iTRAQ-based quantitative proteomic analysis reveals proteomic changes in three fenoxaprop-P-ethyl-resistant Beckmannia syzigachne biotypes with differing ACCase mutations.

PubMed

Pan, Lang; Zhang, Jian; Wang, Junzhi; Yu, Qin; Bai, Lianyang; Dong, Liyao

2017-05-08

American sloughgrass (Beckmannia syzigachne Steud.) is a weed widely distributed in wheat fields of China. In recent years, the evolution of herbicide (fenoxaprop-P-ethyl)-resistant populations has decreased the susceptibility of B. syzigachne. This study compared 4 B. syzigachne populations (3 resistant and 1 susceptible) using iTRAQ to characterize fenoxaprop-P-ethyl resistance in B. syzigachne at the proteomic level. Through searching the UniProt database, 3104 protein species were identified from 13,335 unique peptides. Approximately 2834 protein species were assigned to 23 functional classifications provided by the COG database. Among these, 2299 protein species were assigned to 125 predicted pathways. The resistant biotype contained 8 protein species that changed in abundance relative to the susceptible biotype; they were involved in photosynthesis, oxidative phosphorylation, and fatty acid biosynthesis pathways. In contrast to previous studies comparing only 1 resistant and 1 susceptible population, our use of 3 fenoxaprop-resistant B. syzigachne populations with different genetic backgrounds minimized irrelevant differential expression and eliminated false positives. Therefore, we could more confidently link the differentially expressed proteins to herbicide resistance. Proteomic analysis demonstrated that fenoxaprop-P-ethyl resistance is associated with photosynthetic capacity, a connection that might be related to the target-site mutations in resistant B. syzigachne. This is the first large-scale proteomics study examining herbicide stress responses in different B. syzigachne biotypes. This study has biological relevance because it is the first to employ proteomic analysis for understanding the mechanisms underlying Beckmannia syzigachne herbicide resistance. The plant is a major weed in China and negatively affects crop yield, but has developed considerable resistance to the most common herbicide, fenoxaprop-P-ethyl. Through comparisons of resistant and sensitive biotypes, our study identified multiple proteins (involved in photosynthesis, oxidative phosphorylation, and fatty acid biosynthesis) that are putatively linked to B. syzigachne herbicide response. This large-scale proteomics study, sorely lacking in weed science, contributes valuable data that can be applied to more fine-tuned analyses on the functions of specific proteins in herbicide resistance. Copyright © 2017 Elsevier B.V. All rights reserved.

Sequence/structural analysis of xylem proteome emphasizes pathogenesis-related proteins, chitinases and β-1, 3-glucanases as key players in grapevine defense against Xylella fastidiosa.

PubMed

Chakraborty, Sandeep; Nascimento, Rafael; Zaini, Paulo A; Gouran, Hossein; Rao, Basuthkar J; Goulart, Luiz R; Dandekar, Abhaya M

2016-01-01

Background. Xylella fastidiosa, the causative agent of various plant diseases including Pierce's disease in the US, and Citrus Variegated Chlorosis in Brazil, remains a continual source of concern and economic losses, especially since almost all commercial varieties are sensitive to this Gammaproteobacteria. Differential expression of proteins in infected tissue is an established methodology to identify key elements involved in plant defense pathways. Methods. In the current work, we developed a methodology named CHURNER that emphasizes relevant protein functions from proteomic data, based on identification of proteins with similar structures that do not necessarily have sequence homology. Such clustering emphasizes protein functions which have multiple copies that are up/down-regulated, and highlights similar proteins which are differentially regulated. As a working example we present proteomic data enumerating differentially expressed proteins in xylem sap from grapevines that were infected with X. fastidiosa. Results. Analysis of this data by CHURNER highlighted pathogenesis related PR-1 proteins, reinforcing this as the foremost protein function in xylem sap involved in the grapevine defense response to X. fastidiosa. β-1, 3-glucanase, which has both anti-microbial and anti-fungal activities, is also up-regulated. Simultaneously, chitinases are found to be both up and down-regulated by CHURNER, and thus the net gain of this protein function loses its significance in the defense response. Discussion. We demonstrate how structural data can be incorporated in the pipeline of proteomic data analysis prior to making inferences on the importance of individual proteins to plant defense mechanisms. We expect CHURNER to be applicable to any proteomic data set.

Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients

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 ApoB gives insights into the pathogenesis of TBM.« less

Proteome Analysis of Liver Cells Expressing a Full- Length Hepatitis C Virus (HCV) Replicon and Biopsy Specimens of Posttransplantation Liver from HCV-Infected Patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jacobs, Jon M.; Diamond, Deborah L.; Chan, Eric Y.

2005-06-01

The development of a reproducible model system for the study of Hepatitis C virus (HCV) infection has the potential to significantly enhance the study of virus-host interactions and provide future direction for modeling the pathogenesis of HCV. While there are studies describing global gene expression changes associated with HCV infection, changes in the proteome have not been characterized. We report the first large scale proteome analysis of the highly permissive Huh-7.5 cell line containing a full length HCV replicon. We detected > 4,400 proteins in this cell line, including HCV replicon proteins, using multidimensional liquid chromatographic (LC) separations coupled tomore » mass spectrometry (MS). The set of Huh-7.5 proteins confidently identified is, to our knowledge, the most comprehensive yet reported for a human cell line. Consistent with the literature, a comparison of Huh-7.5 cells (+) and (-) the HCV replicon identified expression changes of proteins involved in lipid metabolism. We extended these analyses to liver biopsy material from HCV-infected patients where > 1,500 proteins were detected from 2 {micro}g protein lysate using the Huh-7.5 protein database and the accurate mass and time (AMT) tag strategy. These findings demonstrate the utility of multidimensional proteome analysis of the HCV replicon model system for assisting the determination of proteins/pathways affected by HCV infection. Our ability to extend these analyses to the highly complex proteome of small liver biopsies with limiting protein yields offers the unique opportunity to begin evaluating the clinical significance of protein expression changes associated with HCV infection.« less

CPTAC Releases Largest-Ever Breast Cancer Proteome Dataset from Previously Genome Characterized Tumors | Office of Cancer Clinical Proteomics Research

Cancer.gov

National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) scientists have released a dataset of proteins and  phosphopeptides identified through deep proteomic and phosphoproteomic analysis of breast tumor samples, previously genomically analyzed by The Cancer Genome Atlas (TCGA).

Mining Missing Membrane Proteins by High-pH Reverse Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry

PubMed Central

Kitata, Reta Birhanu; Dimayacyac-Esleta, Baby Rorielyn T.; Choong, Wai-Kok; Tsai, Chia-Feng; Lin, Tai-Du; Tsou, Chih-Chiang; Weng, Shao-Hsing; Chen, Yi-Ju; Yang, Pan-Chyr; Arco, Susan D.; Nesvizhskii, Alexey I.; Sung, Ting-Yi; Chen, Yu-Ju

2016-01-01

Despite significant efforts in the past decade towards complete mapping of the human proteome, 3564 proteins (neXtProt, 09-2014) are still “missing proteins”. Over one-third of these missing proteins are annotated as membrane proteins, owing to their relatively challenging accessibility with standard shotgun proteomics. Using non-small cell lung cancer (NSCLC) as a model study, we aim to mine missing proteins from disease-associated membrane proteome, which may be still largely under-represented. To increase identification coverage, we employed Hp-RP StageTip pre-fractionation of membrane-enriched samples from 11 NSCLC cell lines. Analysis of membrane samples from 20 pairs of tumor and adjacent normal lung tissue were incorporated to include physiologically expressed membrane proteins. Using multiple search engines (X!Tandem, Comet and Mascot) and stringent evaluation of FDR (MAYU and PeptideShaker), we identified 7702 proteins (66% membrane proteins) and 178 missing proteins (74 membrane proteins) with PSM-, peptide-, and protein-level FDR of 1%. Through multiple reaction monitoring (MRM) using synthetic peptides, we provided additional evidences for 8 missing proteins including 7 with transmembrane helix domains (TMH). This study demonstrates that mining missing proteins focused on cancer membrane sub-proteome can greatly contribute to map the whole human proteome. All data were deposited into ProteomeXchange with the identifier PXD002224. PMID:26202522

CPTAC Releases Largest-Ever Ovarian Cancer Proteome Dataset from Previously Genome Characterized Tumors | Office of Cancer Clinical Proteomics Research

Cancer.gov

National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) scientists have just released a comprehensive dataset of the proteomic analysis of high grade serous ovarian tumor samples, previously genomically analyzed by The Cancer Genome Atlas (TCGA).  This is one of the largest public datasets covering the proteome, phosphoproteome and glycoproteome with complementary deep genomic sequencing data on the same tumor.

The depressed central carbon and energy metabolisms is associated to the acquisition of levofloxacin resistance in Vibrio alginolyticus.

PubMed

Cheng, Zhi-Xue; Yang, Man-Jun; Peng, Bo; Peng, Xuan-Xian; Lin, Xiang-Min; Li, Hui

2018-06-15

The overuse and misuse of antibiotics lead to bacterial antibiotic resistance, challenging human health and intensive cultivation. It is especially required to understand for the mechanism of antibiotic resistance to control antibiotic-resistant pathogens. The present study characterized the differential proteome of levofloxacin-resistant Vibrio alginolyticus with the most advanced iTRAQ quantitative proteomics technology. A total of 160 proteins of differential abundance were identified, where 70 were decreased and 90 were increased. Further analysis demonstrated that crucial metabolic pathways like TCA cycle were significantly down-regulated. qRT-PCR analysis demonstrated the decreased gene expression of glycolysis/gluconeogenesis, the TCA cycle, and fatty acid biosynthesis. Moreover, Na(+)-NQR complex gene expression, membrane potential and the adenylate energy charge ratio were decreased, indicating that the decreased central carbon metabolism is associated to the acquisition of levofloxacin resistance. Therefore, the reduced central carbon and energy metabolisms form a characteristic feature as fitness costs of V. alginolyticus in resistance to levofloxacin. The overuse and misuse of antibiotics lead to bacterial antibiotic resistance, challenging human health and intensive cultivation. Understanding for the antibiotic resistance mechanisms is especially required to control these antibiotic-resistant pathogens. The present study characterized the differential proteome of levofloxacin-resistant Vibrio alginolyticus using the most advanced iTRAQ quantitative proteomics technology. A total of 160 differential abundance of proteins were identified with 70 decreases and 90 increases by liquid chromatography matrix assisted laser desorption ionization mass spectrometry. Most interestingly, crucial metabolic pathways such as the TCA cycle sharply fluctuated. This is the first report that the reduced central carbon and energy metabolisms form a characteristic feature as a mechanism of V. alginolyticus in resistance to levofloxacin. Copyright © 2018 Elsevier B.V. All rights reserved.

Analysis by two-dimensional Blue Native/SDS-PAGE of membrane protein alterations in rat soleus muscle after hindlimb unloading.

PubMed

Basco, Davide; Nicchia, Grazia Paola; Desaphy, Jean-François; Camerino, Diana Conte; Frigeri, Antonio; Svelto, Maria

2010-12-01

Muscle atrophy occurring in several pathophysiological conditions determines decreases in muscle protein synthesis, increases in the rate of proteolysis and changes in muscle fiber composition. To determine the effect of muscle atrophy induced by hindlimb unloading (HU) on membrane proteins from rat soleus, a proteomic approach based on two-dimensional Blue Native/SDS-PAGE was performed. Proteomic analysis of normal and HU soleus muscle demonstrates statistically significant changes in the relative level of 36 proteins. Among the proteins identified by mass spectrometry, most are involved in pathways associated with muscle fuel utilization, indicating a shift in metabolism from oxidative to glycolytic. Moreover, immunoblotting analysis revealed an increase in aquaporin-4 (AQP4) water channel and an alteration of proteins belonging to the dystrophin-glycoprotein complex (DGC). AQP4 and DGC are regulated in soleus muscle subjected to simulated microgravity in response to compensatory mechanisms induced by muscle atrophy, and they parallel the slow-to-fast twitch conversion that occurs in soleus fibers during HU. In conclusion, the alterations of soleus muscle membrane proteome may play a pivotal role in the mechanisms involved in disuse-induced muscle atrophy.

A novel proteomics sample preparation method for secretome analysis of Hypocrea jecorina growing on insoluble substrates.

PubMed

Bengtsson, Oskar; Arntzen, Magnus Ø; Mathiesen, Geir; Skaugen, Morten; Eijsink, Vincent G H

2016-01-10

Analysis of the secretomes of filamentous fungi growing on insoluble lignocellulosic substrates is of major current interest because of the industrial potential of secreted fungal enzymes. Importantly, such studies can help identifying key enzymes from a large arsenal of bioinformatically detected candidates in fungal genomes. We describe a simple, plate-based method to analyze the secretome of Hypocrea jecorina growing on insoluble substrates that allows harsh sample preparation methods promoting desorption, and subsequent identification, of substrate-bound proteins, while minimizing contamination with non-secreted proteins from leaking or lysed cells. The validity of the method was demonstrated by comparative secretome analysis of wild-type H.jecorina strain QM6a growing on bagasse, birch wood, spruce wood or pure cellulose, using label-fee quantification. The proteomic data thus obtained were consistent with existing data from transcriptomics and proteomics studies and revealed clear differences in the responses to complex lignocellulosic substrates and the response to pure cellulose. This easy method is likely to be generally applicable to filamentous fungi and to other microorganisms growing on insoluble substrates. Copyright © 2015 Elsevier B.V. All rights reserved.

A proteomic analysis identifies candidate early biomarkers to predict ovarian hyperstimulation syndrome in polycystic ovarian syndrome patients.

PubMed

Wu, Lan; Sun, Yazhou; Wan, Jun; Luan, Ting; Cheng, Qing; Tan, Yong

2017-07-01

Ovarian hyperstimulation syndrome (OHSS) is a potentially life‑threatening, iatrogenic complication that occurs during assisted reproduction. Polycystic ovarian syndrome (PCOS) significantly increases the risk of OHSS during controlled ovarian stimulation. Therefore, a more effective early prediction technique is required in PCOS patients. Quantitative proteomic analysis of serum proteins indicates the potential diagnostic value for disease. In the present study, the authors revealed the differentially expressed proteins in OHSS patients with PCOS as new diagnostic biomarkers. The promising proteins obtained from liquid chromatography‑mass spectrometry were subjected to ELISA and western blotting assay for further confirmation. A total of 57 proteins were identified with significant difference, of which 29 proteins were upregulated and 28 proteins were downregulated in OHSS patients. Haptoglobin, fibrinogen and lipoprotein lipase were selected as candidate biomarkers. Receiver operating characteristic curve analysis demonstrated all three proteins may have potential as biomarkers to discriminate OHSS in PCOS patients. Haptoglobin, fibrinogen and lipoprotein lipase have never been reported as a predictive marker of OHSS in PCOS patients, and their potential roles in OHSS occurrence deserve further studies. The proteomic results reported in the present study may gain deeper insights into the pathophysiology of OHSS.

Low Cost, Scalable Proteomics Data Analysis Using Amazon's Cloud Computing Services and Open Source Search Algorithms

PubMed Central

Halligan, Brian D.; Geiger, Joey F.; Vallejos, Andrew K.; Greene, Andrew S.; Twigger, Simon N.

2009-01-01

One of the major difficulties for many laboratories setting up proteomics programs has been obtaining and maintaining the computational infrastructure required for the analysis of the large flow of proteomics data. We describe a system that combines distributed cloud computing and open source software to allow laboratories to set up scalable virtual proteomics analysis clusters without the investment in computational hardware or software licensing fees. Additionally, the pricing structure of distributed computing providers, such as Amazon Web Services, allows laboratories or even individuals to have large-scale computational resources at their disposal at a very low cost per run. We provide detailed step by step instructions on how to implement the virtual proteomics analysis clusters as well as a list of current available preconfigured Amazon machine images containing the OMSSA and X!Tandem search algorithms and sequence databases on the Medical College of Wisconsin Proteomics Center website (http://proteomics.mcw.edu/vipdac). PMID:19358578

Low cost, scalable proteomics data analysis using Amazon's cloud computing services and open source search algorithms.

PubMed

Halligan, Brian D; Geiger, Joey F; Vallejos, Andrew K; Greene, Andrew S; Twigger, Simon N

2009-06-01

One of the major difficulties for many laboratories setting up proteomics programs has been obtaining and maintaining the computational infrastructure required for the analysis of the large flow of proteomics data. We describe a system that combines distributed cloud computing and open source software to allow laboratories to set up scalable virtual proteomics analysis clusters without the investment in computational hardware or software licensing fees. Additionally, the pricing structure of distributed computing providers, such as Amazon Web Services, allows laboratories or even individuals to have large-scale computational resources at their disposal at a very low cost per run. We provide detailed step-by-step instructions on how to implement the virtual proteomics analysis clusters as well as a list of current available preconfigured Amazon machine images containing the OMSSA and X!Tandem search algorithms and sequence databases on the Medical College of Wisconsin Proteomics Center Web site ( http://proteomics.mcw.edu/vipdac ).

PeptideDepot: Flexible Relational Database for Visual Analysis of Quantitative Proteomic Data and Integration of Existing Protein Information

PubMed Central

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

New Funding Opportunity Announcements (FOAs): Reissuance of Clinical Proteomic Tumor Analysis Consortium (CPTAC) | Office of Cancer Clinical Proteomics Research

Cancer.gov

The National Cancer Institute is soliciting applications for the reissuance of its Clinical Proteomic Tumor Analysis Consortium (CPTAC) program.   CPTAC will support broad efforts focused on several cancer types to explore further the complexities of cancer proteomes and their connections to abnormalities in cancer genomes.

A novel quantification-driven proteomic strategy identifies an endogenous peptide of pleiotrophin as a new biomarker of Alzheimer's disease.

PubMed

Skillbäck, Tobias; Mattsson, Niklas; Hansson, Karl; Mirgorodskaya, Ekaterina; Dahlén, Rahil; van der Flier, Wiesje; Scheltens, Philip; Duits, Floor; Hansson, Oskar; Teunissen, Charlotte; Blennow, Kaj; Zetterberg, Henrik; Gobom, Johan

2017-10-17

We present a new, quantification-driven proteomic approach to identifying biomarkers. In contrast to the identification-driven approach, limited in scope to peptides that are identified by database searching in the first step, all MS data are considered to select biomarker candidates. The endopeptidome of cerebrospinal fluid from 40 Alzheimer's disease (AD) patients, 40 subjects with mild cognitive impairment, and 40 controls with subjective cognitive decline was analyzed using multiplex isobaric labeling. Spectral clustering was used to match MS/MS spectra. The top biomarker candidate cluster (215% higher in AD compared to controls, area under ROC curve = 0.96) was identified as a fragment of pleiotrophin located near the protein's C-terminus. Analysis of another cohort (n = 60 over four clinical groups) verified that the biomarker was increased in AD patients while no change in controls, Parkinson's disease or progressive supranuclear palsy was observed. The identification of the novel biomarker pleiotrophin 151-166 demonstrates that our quantification-driven proteomic approach is a promising method for biomarker discovery, which may be universally applicable in clinical proteomics.

Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-Based Untargeted Quantitative Proteomic Approach To Identify Change of the Plasma Proteins by Salbutamol Abuse in Beef Cattle.

PubMed

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.

Proteome Analysis of the Plasma Membrane of Mycobacterium Tuberculosis

PubMed Central

Arora, Shalini; Kosalai, K.; Namane, Abdelkader; Pym, Alex S.; Cole, Stewart T.

2002-01-01

The plasma membrane of Mycobacterium tuberculosis is likely to contain proteins that could serve as novel drug targets, diagnostic probes or even components of a vaccine against tuberculosis. With this in mind, we have undertaken proteome analysis of the membrane of M. tuberculosis H37Rv. Isolated membrane vesicles were extracted with either a detergent (Triton X114) or an alkaline buffer (carbonate) following two of the protocols recommended for membrane protein enrichment. Proteins were resolved by 2D-GE using immobilized pH gradient (IPG) strips, and identified by peptide mass mapping utilizing the M. tuberculosis genome database. The two extraction procedures yielded patterns with minimal overlap. Only two proteins, both HSPs, showed a common presence. MALDI–MS analysis of 61 spots led to the identification of 32 proteins, 17 of which were new to the M. tuberculosis proteome database. We classified 19 of the identified proteins as ‘membrane-associated’; 14 of these were further classified as ‘membrane-bound’, three of which were lipoproteins. The remaining proteins included four heat-shock proteins and several enzymes involved in energy or lipid metabolism. Extraction with Triton X114 was found to be more effective than carbonate for detecting ‘putative’ M. tuberculosis membrane proteins. The protocol was also found to be suitable for comparing BCG and M. tuberculosis membranes, identifying ESAT-6 as being expressed selectively in M. tuberculosis. While this study demonstrates for the first time some of the membrane proteins of M. tuberculosis, it also underscores the problems associated with proteomic analysis of a complex membrane such as that of a mycobacterium. PMID:18629250

Evolutionary conservation of the polyproline II conformation surrounding intrinsically disordered phosphorylation sites.

PubMed

Elam, W Austin; Schrank, Travis P; Campagnolo, Andrew J; Hilser, Vincent J

2013-04-01

Intrinsically disordered (ID) proteins function in the absence of a unique stable structure and appear to challenge the classic structure-function paradigm. The extent to which ID proteins take advantage of subtle conformational biases to perform functions, and whether signals for such mechanism can be identified in proteome-wide studies is not well understood. Of particular interest is the polyproline II (PII) conformation, suggested to be highly populated in unfolded proteins. We experimentally determine a complete calorimetric propensity scale for the PII conformation. Projection of the scale into representative eukaryotic proteomes reveals significant PII bias in regions coding for ID proteins. Importantly, enrichment of PII in ID proteins, or protein segments, is also captured by other PII scales, indicating that this enrichment is robustly encoded and universally detectable regardless of the method of PII propensity determination. Gene ontology (GO) terms obtained using our PII scale and other scales demonstrate a consensus for molecular functions performed by high PII proteins across the proteome. Perhaps the most striking result of the GO analysis is conserved enrichment (P < 10(-8) ) of phosphorylation sites in high PII regions found by all PII scales. Subsequent conformational analysis reveals a phosphorylation-dependent modulation of PII, suggestive of a conserved "tunability" within these regions. In summary, the application of an experimentally determined polyproline II (PII) propensity scale to proteome-wide sequence analysis and gene ontology reveals an enrichment of PII bias near disordered phosphorylation sites that is conserved throughout eukaryotes. Copyright © 2013 The Protein Society.

Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to Multidimensional Protein Identification Technology

PubMed Central

Gonzalez-Begne, Mireya; Lu, Bingwen; Liao, Lujian; Xu, Tao; Bedi, Gurrinder; Melvin, James E.; Yates, John R.

2011-01-01

In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases. PMID:21936497

The broccoli (Brassica oleracea) phloem tissue proteome.

PubMed

Anstead, James A; Hartson, Steven D; Thompson, Gary A

2013-11-07

The transport of sugars, hormones, amino acids, proteins, sugar alcohols, and other organic compounds from the sites of synthesis to the sites of use or storage occurs through the conducting cells of the phloem. To better understand these processes a comprehensive understanding of the proteins involved is required. While a considerable amount of data has been obtained from proteomic analyses of phloem sap, this has mainly served to identify the soluble proteins that are translocated through the phloem network. In order to obtain more comprehensive proteomic data from phloem tissue we developed a simple dissection procedure to isolate phloem tissue from Brassica oleracea. The presence of a high density of phloem sieve elements was confirmed using light microscopy and fluorescently labeled sieve element-specific antibodies. To increase the depth of the proteomic analysis for membrane bound and associated proteins, soluble proteins were extracted first and subsequent extractions were carried out using two different detergents (SDS and CHAPSO). Across all three extractions almost four hundred proteins were identified and each extraction method added to the analysis demonstrating the utility of an approach combining several extraction protocols. The phloem was found to be enriched in proteins associated with biotic and abiotic stress responses and structural proteins. Subsequent expression analysis identified a number of genes that appear to be expressed exclusively or at very high levels in phloem tissue, including genes that are known to express specifically in the phloem as well as novel phloem genes.

Quantitative proteomics reveals ecological fitness cost of multi-herbicide resistant barnyardgrass (Echinochloa crus-galli L.).

PubMed

Yang, Xia; Zhang, Zichang; Gu, Tao; Dong, Mingchao; Peng, Qiong; Bai, Lianyang; Li, Yongfeng

2017-01-06

Barnyardgrass (Echinochloa crus-galli) is one of the top 15 herbicide-resistant weeds around the world that interferes with rice growth, resulting in major losses of rice yield. Thus, multi-herbicide resistance in barnyardgrass presents a major threat, with the underlying mechanisms that contribute to resistance requiring elucidation. In an attempt to characterize this multi-herbicide resistance at the proteomic level, comparative analysis of resistant and susceptible barnyardgrasses was performed using iTRAQ, both with and without quinclorac, bispyribac-sodium and penoxsulam herbicidal treatment. A total of 1342 protein species were identified from 2248 unique peptides by searching the UniProt database and conducting data analysis. Approximately 904 protein species with 4774 Gene Ontology (GO) terms were grouped into the categories of biological process, cellular component and molecular function. Among these, 688 protein species were annotated into 1583 KEGG pathways, with 980 protein species relating to metabolism and 93 relating to environmental information processing. A total of 292 protein species showed more than a 1.2-fold change in abundance in the resistant biotype relative to the susceptible biotype. Furthermore, herbicide treatment resulted in 157 protein species that showed more than a 1.2-fold change in the resistant biotype. Moreover, physiological analyses demonstrated an ecological fitness cost in the resistant biotype. While some studies have shown a fitness cost to be associated with an altered ecological interaction, our understanding of the fitness costs associated with herbicide resistance are limited. Herein, physiological and proteomic analysis demonstrates herbicide resistance associated ecological fitness cost and potential mechanisms of herbicide-resistance in resistant biotypes of E. crus-galli. The results presented herein have revealed differences in ecological adaptation between resistant and susceptible biotypes in E. crus-galli and provide a fundamental basis enabling the development of new strategies for weed control. Lastly, this is the first large-scale proteomics study to examine herbicide stress responses in different barnyardgrass biotypes. Copyright © 2016 Elsevier B.V. All rights reserved.

Long-term heat stress induces the inflammatory response in dairy cows revealed by plasma proteome analysis.

PubMed

Min, Li; Zheng, Nan; Zhao, Shengguo; Cheng, Jianbo; Yang, Yongxin; Zhang, Yangdong; Yang, Hongjian; Wang, Jiaqi

2016-03-04

In this work we employed a comparative proteomic approach to evaluate seasonal heat stress and investigate proteomic alterations in plasma of dairy cows. Twelve lactating Holstein dairy cows were used and the treatments were: heat stress (n = 6) in hot summer (at the beginning of the moderate heat stress) and no heat stress (n = 6) in spring natural ambient environment, respectively. Subsequently, heat stress treatment lasted 23 days (at the end of the moderate heat stress) to investigate the alterations of plasma proteins, which might be employed as long-term moderate heat stress response in dairy cows. Changes in plasma proteins were analyzed by two-dimensional electrophoresis (2-DE) combined with mass spectrometry. Analysis of the properties of the identified proteins revealed that the alterations of plasma proteins were related to inflammation in long-term moderate heat stress. Furthermore, the increase in plasma tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) directly demonstrated that long-term moderate heat stress caused an inflammatory response in dairy cows. Copyright © 2016 Elsevier Inc. All rights reserved.

Proteomic Analysis of Laser Microdissected Melanoma Cells from Skin Organ Cultures

PubMed Central

Hood, Brian L.; Grahovac, Jelena; Flint, Melanie S.; Sun, Mai; Charro, Nuno; Becker, Dorothea; Wells, Alan; Conrads, Thomas P

2010-01-01

Gaining insights into the molecular events that govern the progression from melanoma in situ to advanced melanoma, and understanding how the local microenvironment at the melanoma site influences this progression, are two clinically pivotal aspects that to date are largely unexplored. In an effort to identify key regulators of the crosstalk between melanoma cells and the melanoma-skin microenvironment, primary and metastatic human melanoma cells were seeded into skin organ cultures (SOCs), and grown for two weeks. Melanoma cells were recovered from SOCs by laser microdissection and whole-cell tryptic digests analyzed by nanoflow liquid chromatography-tandem mass spectrometry with an LTQ-Orbitrap. The differential protein abundances were calculated by spectral counting, the results of which provides evidence that cell-matrix and cell-adhesion molecules that are upregulated in the presence of these melanoma cells recapitulate proteomic data obtained from comparative analysis of human biopsies of invasive melanoma and a tissue sample of adjacent, non-involved skin. This concordance demonstrates the value of SOCs for conducting proteomic investigations of the melanoma microenvironment. PMID:20459140

Proteomic Investigation of Aphid Honeydew Reveals an Unexpected Diversity of Proteins

PubMed Central

Haubruge, Eric; Hance, Thierry; Thonart, Philippe; De Pauw, Edwin; Francis, Frédéric

2013-01-01

Aphids feed on the phloem sap of plants, and are the most common honeydew-producing insects. While aphid honeydew is primarily considered to comprise sugars and amino acids, its protein diversity has yet to be documented. Here, we report on the investigation of the honeydew proteome from the pea aphid Acyrthosiphon pisum. Using a two-Dimensional Differential in-Gel Electrophoresis (2D-Dige) approach, more than 140 spots were isolated, demonstrating that aphid honeydew also represents a diverse source of proteins. About 66% of the isolated spots were identified through mass spectrometry analysis, revealing that the protein diversity of aphid honeydew originates from several organisms (i.e. the host aphid and its microbiota, including endosymbiotic bacteria and gut flora). Interestingly, our experiments also allowed to identify some proteins like chaperonin, GroEL and Dnak chaperones, elongation factor Tu (EF-Tu), and flagellin that might act as mediators in the plant-aphid interaction. In addition to providing the first aphid honeydew proteome analysis, we propose to reconsider the importance of this substance, mainly acknowledged to be a waste product, from the aphid ecology perspective. PMID:24086359

PSEA-Quant: a protein set enrichment analysis on label-free and label-based protein quantification data.

PubMed

Lavallée-Adam, Mathieu; Rauniyar, Navin; McClatchy, Daniel B; Yates, John R

2014-12-05

The majority of large-scale proteomics quantification methods yield long lists of quantified proteins that are often difficult to interpret and poorly reproduced. Computational approaches are required to analyze such intricate quantitative proteomics data sets. We propose a statistical approach to computationally identify protein sets (e.g., Gene Ontology (GO) terms) that are significantly enriched with abundant proteins with reproducible quantification measurements across a set of replicates. To this end, we developed PSEA-Quant, a protein set enrichment analysis algorithm for label-free and label-based protein quantification data sets. It offers an alternative approach to classic GO analyses, models protein annotation biases, and allows the analysis of samples originating from a single condition, unlike analogous approaches such as GSEA and PSEA. We demonstrate that PSEA-Quant produces results complementary to GO analyses. We also show that PSEA-Quant provides valuable information about the biological processes involved in cystic fibrosis using label-free protein quantification of a cell line expressing a CFTR mutant. Finally, PSEA-Quant highlights the differences in the mechanisms taking place in the human, rat, and mouse brain frontal cortices based on tandem mass tag quantification. Our approach, which is available online, will thus improve the analysis of proteomics quantification data sets by providing meaningful biological insights.

PSEA-Quant: A Protein Set Enrichment Analysis on Label-Free and Label-Based Protein Quantification Data

PubMed Central

2015-01-01

The majority of large-scale proteomics quantification methods yield long lists of quantified proteins that are often difficult to interpret and poorly reproduced. Computational approaches are required to analyze such intricate quantitative proteomics data sets. We propose a statistical approach to computationally identify protein sets (e.g., Gene Ontology (GO) terms) that are significantly enriched with abundant proteins with reproducible quantification measurements across a set of replicates. To this end, we developed PSEA-Quant, a protein set enrichment analysis algorithm for label-free and label-based protein quantification data sets. It offers an alternative approach to classic GO analyses, models protein annotation biases, and allows the analysis of samples originating from a single condition, unlike analogous approaches such as GSEA and PSEA. We demonstrate that PSEA-Quant produces results complementary to GO analyses. We also show that PSEA-Quant provides valuable information about the biological processes involved in cystic fibrosis using label-free protein quantification of a cell line expressing a CFTR mutant. Finally, PSEA-Quant highlights the differences in the mechanisms taking place in the human, rat, and mouse brain frontal cortices based on tandem mass tag quantification. Our approach, which is available online, will thus improve the analysis of proteomics quantification data sets by providing meaningful biological insights. PMID:25177766

Evaluation of Selected Binding Domains for the Analysis of Ubiquitinated Proteomes

NASA Astrophysics Data System (ADS)

Nakayasu, Ernesto S.; Ansong, Charles; Brown, Joseph N.; Yang, Feng; Lopez-Ferrer, Daniel; Qian, Wei-Jun; Smith, Richard D.; Adkins, Joshua N.

2013-08-01

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono- and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ~200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle.

Comparative Proteomic Analysis of Liver Steatosis and Fibrosis after Oral Hepatotoxicant Administration in Sprague-Dawley Rats.

PubMed

McDyre, B Claire; AbdulHameed, Mohamed Diwan M; Permenter, Matthew G; Dennis, William E; Baer, Christine E; Koontz, Jason M; Boyle, Molly H; Wallqvist, Anders; Lewis, John A; Ippolito, Danielle L

2018-02-01

The past decade has seen an increase in the development and clinical use of biomarkers associated with histological features of liver disease. Here, we conduct a comparative histological and global proteomics analysis to identify coregulated modules of proteins in the progression of hepatic steatosis or fibrosis. We orally administered the reference chemicals bromobenzene (BB) or 4,4'-methylenedianiline (4,4'-MDA) to male Sprague-Dawley rats for either 1 single administration or 5 consecutive daily doses. Livers were preserved for histopathology and global proteomics assessment. Analysis of liver sections confirmed a dose- and time-dependent increase in frequency and severity of histopathological features indicative of lipid accumulation after BB or fibrosis after 4,4'-MDA. BB administration resulted in a dose-dependent increase in the frequency and severity of inflammation and vacuolation. 4,4'-MDA administration resulted in a dose-dependent increase in the frequency and severity of periportal collagen accumulation and inflammation. Pathway analysis identified a time-dependent enrichment of biological processes associated with steatogenic or fibrogenic initiating events, cellular functions, and toxicological states. Differentially expressed protein modules were consistent with the observed histology, placing physiologically linked protein networks into context of the disease process. This study demonstrates the potential for protein modules to provide mechanistic links between initiating events and histopathological outcomes.

Data for a proteomic analysis of p53-independent induction of apoptosis by bortezomib

PubMed Central

Yerlikaya, Azmi; Okur, Emrah; Tarık Baykal, Ahmet; Acılan, Ceyda; Boyacı, İhsan; Ulukaya, Engin

2014-01-01

This data article contains data related to the research article entitled, “A proteomic analysis of p53-independent induction of apoptosis by bortezomib in 4T1 breast cancer cell line” by Yerlikaya et al. [1]. The research article presented 2-DE and nLC-MS/MS based proteomic analysis of proteasome inhibitor bortezomib-induced changes in the expression of cellular proteins. The report showed that GRP78 and TCEB2 were over-expressed in response to treatment with bortezomib for 24 h. In addition, the report demonstrated that Hsp70, the 26S proteasome non-ATPase regulatory subunit 14 and sequestosome 1 were increased at least 2 fold in p53-deficient 4T1 cells. The data here show for the first time the increased expressions of Card10, Dffb, Traf3 and Trp53bp2 in response to inhibition of the 26S proteasome. The information presented here also shows that both Traf1 and Xiap (a member of IAPs) are also downregulated simultaneously upon proteasomal inhibition. The increases in the level of Card10 and Trp53bp2 proteins were verified by Western blot analysis in response to varying concentrations of bortezomib for 24 h. PMID:26217687

A Comparison of Protein Extraction Methods Suitable for Gel-Based Proteomic Studies of Aphid Proteins

PubMed Central

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

Matrilin-1 expression is increased in the vertebral column of Atlantic salmon (Salmo salar L.) individuals displaying spinal fusions.

PubMed

Pedersen, Mona E; Takle, Harald; Ytteborg, Elisabeth; Veiseth-Kent, Eva; Enersen, Grethe; Færgestad, Ellen; Baeverfjord, Grete; Hannesson, Kirsten O

2011-12-01

We have previously characterized the development of vertebral fusions induced by elevated water temperature in Atlantic salmon. Molecular markers of bone and cartilage development together with histology were used to understand the complex pathology and mechanism in the development of this spinal malformation. In this study, we wanted to use proteomics, a non-hypothetical approach to screen for possible new markers involved in the fusion process. Proteins extracted from non-deformed and fused vertebrae of Atlantic salmon were therefore compared by two-dimensional electrophoresis (2DE) and MALDI-TOF analysis. Data analysis of protein spots in the 2DE gels demonstrated matrilin-1, also named cartilage matrix protein, to be the most highly up-regulated protein in fused compared with non-deformed vertebrae. Furthermore, real-time PCR analysis showed strong up-regulation of matrilin-1 mRNA in fused vertebrae. Immunohistochemistry demonstrated induced matrilin-1 expression in trans-differentiating cells undergoing a metaplastic shift toward chondrocytes in fusing vertebrae, whereas abundant expression was demonstrated in cartilaginous tissue and chordocytes of both non-deformed and fused vertebrae. These results identifies matrilin-1 as a new interesting candidate in the fusion process, and ratify the use of proteomic as a valuable technique to screen for markers involved in vertebral pathogenesis.

CPTAC | Office of Cancer Clinical Proteomics Research

Cancer.gov

The National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) is a national effort to accelerate the understanding of the molecular basis of cancer through the application of large-scale proteome and genome analysis, or proteogenomics.

A software suite for the generation and comparison of peptide arrays from sets of data collected by liquid chromatography-mass spectrometry.

PubMed

Li, Xiao-jun; Yi, Eugene C; Kemp, Christopher J; Zhang, Hui; Aebersold, Ruedi

2005-09-01

There is an increasing interest in the quantitative proteomic measurement of the protein contents of substantially similar biological samples, e.g. for the analysis of cellular response to perturbations over time or for the discovery of protein biomarkers from clinical samples. Technical limitations of current proteomic platforms such as limited reproducibility and low throughput make this a challenging task. A new LC-MS-based platform is able to generate complex peptide patterns from the analysis of proteolyzed protein samples at high throughput and represents a promising approach for quantitative proteomics. A crucial component of the LC-MS approach is the accurate evaluation of the abundance of detected peptides over many samples and the identification of peptide features that can stratify samples with respect to their genetic, physiological, or environmental origins. We present here a new software suite, SpecArray, that generates a peptide versus sample array from a set of LC-MS data. A peptide array stores the relative abundance of thousands of peptide features in many samples and is in a format identical to that of a gene expression microarray. A peptide array can be subjected to an unsupervised clustering analysis to stratify samples or to a discriminant analysis to identify discriminatory peptide features. We applied the SpecArray to analyze two sets of LC-MS data: one was from four repeat LC-MS analyses of the same glycopeptide sample, and another was from LC-MS analysis of serum samples of five male and five female mice. We demonstrate through these two study cases that the SpecArray software suite can serve as an effective software platform in the LC-MS approach for quantitative proteomics.

Mining Missing Membrane Proteins by High-pH Reverse-Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry.

PubMed

Kitata, Reta Birhanu; Dimayacyac-Esleta, Baby Rorielyn T; Choong, Wai-Kok; Tsai, Chia-Feng; Lin, Tai-Du; Tsou, Chih-Chiang; Weng, Shao-Hsing; Chen, Yi-Ju; Yang, Pan-Chyr; Arco, Susan D; Nesvizhskii, Alexey I; Sung, Ting-Yi; Chen, Yu-Ju

2015-09-04

Despite significant efforts in the past decade toward complete mapping of the human proteome, 3564 proteins (neXtProt, 09-2014) are still "missing proteins". Over one-third of these missing proteins are annotated as membrane proteins, owing to their relatively challenging accessibility with standard shotgun proteomics. Using nonsmall cell lung cancer (NSCLC) as a model study, we aim to mine missing proteins from disease-associated membrane proteome, which may be still largely under-represented. To increase identification coverage, we employed Hp-RP StageTip prefractionation of membrane-enriched samples from 11 NSCLC cell lines. Analysis of membrane samples from 20 pairs of tumor and adjacent normal lung tissue was incorporated to include physiologically expressed membrane proteins. Using multiple search engines (X!Tandem, Comet, and Mascot) and stringent evaluation of FDR (MAYU and PeptideShaker), we identified 7702 proteins (66% membrane proteins) and 178 missing proteins (74 membrane proteins) with PSM-, peptide-, and protein-level FDR of 1%. Through multiple reaction monitoring using synthetic peptides, we provided additional evidence of eight missing proteins including seven with transmembrane helix domains. This study demonstrates that mining missing proteins focused on cancer membrane subproteome can greatly contribute to map the whole human proteome. All data were deposited into ProteomeXchange with the identifier PXD002224.

Suberoylanilide hydroxamic acid treatment reveals crosstalks among proteome, ubiquitylome and acetylome in non-small cell lung cancer A549 cell line.

PubMed

Wu, Quan; Cheng, Zhongyi; Zhu, Jun; Xu, Weiqing; Peng, Xiaojun; Chen, Chuangbin; Li, Wenting; Wang, Fengsong; Cao, Lejie; Yi, Xingling; Wu, Zhiwei; Li, Jing; Fan, Pingsheng

2015-03-31

Suberoylanilide hydroxamic acid (SAHA) is a well-known histone deacetylase (HDAC) inhibitor and has been used as practical therapy for breast cancer and non-small cell lung cancer (NSCLC). It is previously demonstrated that SAHA treatment could extensively change the profile of acetylome and proteome in cancer cells. However, little is known about the impact of SAHA on other protein modifications and the crosstalks among different modifications and proteome, hindering the deep understanding of SAHA-mediated cancer therapy. In this work, by using SILAC technique, antibody-based affinity enrichment and high-resolution LC-MS/MS analysis, we investigated quantitative proteome, acetylome and ubiquitylome as well as crosstalks among the three datasets in A549 cells toward SAHA treatment. In total, 2968 proteins, 1099 acetylation sites and 1012 ubiquitination sites were quantified in response to SAHA treatment, respectively. With the aid of intensive bioinformatics, we revealed that the proteome and ubiquitylome were negatively related upon SAHA treatment. Moreover, the impact of SAHA on acetylome resulted in 258 up-regulated and 99 down-regulated acetylation sites at the threshold of 1.5 folds. Finally, we identified 55 common sites with both acetylation and ubiquitination, among which ubiquitination level in 43 sites (78.2%) was positive related to acetylation level.

Global Analysis of Salmonella Alternative Sigma Factor E on Protein Translation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jie; Nakayasu, Ernesto S.; Overall, Christopher C.

The alternative sigma factor E (σ E) is critical for response to extracytoplasmic stress in Salmonella. Extensive studies have been conducted on σ E-regulated gene expression, particularly at the transcriptional level. Increasing evidence suggests however that σ E may indirectly participate in post-transcriptional regulation. Here in this study, we conducted sample-matched global proteomic and transcriptomic analyses to determine the level of regulation mediated by σ E in Salmonella. We analysed samples from wild type and isogenic rpoE mutant Salmonella cultivated in three different conditions; nutrient-rich and conditions that mimic early and late intracellular infection. We found that 30% of themore » observed proteome was regulated by σ E combining all three conditions. In different growth conditions, σ E affected the expression of a broad spectrum of Salmonella proteins required for miscellaneous functions. Those involved in transport and binding, protein synthesis, and stress response were particularly highlighted. By comparing transcriptomic and proteomic data, we identified genes post-transcriptionally regulated by σ E and found that post-transcriptional regulation was responsible for a majority of changes observed in the σ E-regulated proteome. Further, comparison of transcriptomic and proteomic data from hfq mutant of Salmonella demonstrated that σ E–mediated post-transcriptional regulation was partially dependent on the RNA-binding protein Hfq.« less

Global Analysis of Salmonella Alternative Sigma Factor E on Protein Translation

DOE PAGES

Li, Jie; Nakayasu, Ernesto S.; Overall, Christopher C.; ...

2015-02-16

The alternative sigma factor E (σ E) is critical for response to extracytoplasmic stress in Salmonella. Extensive studies have been conducted on σ E-regulated gene expression, particularly at the transcriptional level. Increasing evidence suggests however that σ E may indirectly participate in post-transcriptional regulation. Here in this study, we conducted sample-matched global proteomic and transcriptomic analyses to determine the level of regulation mediated by σ E in Salmonella. We analysed samples from wild type and isogenic rpoE mutant Salmonella cultivated in three different conditions; nutrient-rich and conditions that mimic early and late intracellular infection. We found that 30% of themore » observed proteome was regulated by σ E combining all three conditions. In different growth conditions, σ E affected the expression of a broad spectrum of Salmonella proteins required for miscellaneous functions. Those involved in transport and binding, protein synthesis, and stress response were particularly highlighted. By comparing transcriptomic and proteomic data, we identified genes post-transcriptionally regulated by σ E and found that post-transcriptional regulation was responsible for a majority of changes observed in the σ E-regulated proteome. Further, comparison of transcriptomic and proteomic data from hfq mutant of Salmonella demonstrated that σ E–mediated post-transcriptional regulation was partially dependent on the RNA-binding protein Hfq.« less

Comparative bioinformatics analyses and profiling of lysosome-related organelle proteomes

NASA Astrophysics Data System (ADS)

Hu, Zhang-Zhi; Valencia, Julio C.; Huang, Hongzhan; Chi, An; Shabanowitz, Jeffrey; Hearing, Vincent J.; Appella, Ettore; Wu, Cathy

2007-01-01

Complete and accurate profiling of cellular organelle proteomes, while challenging, is important for the understanding of detailed cellular processes at the organelle level. Mass spectrometry technologies coupled with bioinformatics analysis provide an effective approach for protein identification and functional interpretation of organelle proteomes. In this study, we have compiled human organelle reference datasets from large-scale proteomic studies and protein databases for seven lysosome-related organelles (LROs), as well as the endoplasmic reticulum and mitochondria, for comparative organelle proteome analysis. Heterogeneous sources of human organelle proteins and rodent homologs are mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. Cataloging organelle proteomes allows close examination of both shared and unique proteins among various LROs and reveals their functional relevance. The proteomic comparisons show that LROs are a closely related family of organelles. The shared proteins indicate the dynamic and hybrid nature of LROs, while the unique transmembrane proteins may represent additional candidate marker proteins for LROs. This comparative analysis, therefore, provides a basis for hypothesis formulation and experimental validation of organelle proteins and their functional roles.

Assessing signal-to-noise in quantitative proteomics: multivariate statistical analysis in DIGE experiments.

PubMed

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.

Quantitative proteomic analysis reveals that antioxidation mechanisms contribute to cold tolerance in plantain (Musa paradisiaca L.; ABB Group) seedlings.

PubMed

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 proteomic analysis.

Quantitative Proteomic Analysis Reveals that Antioxidation Mechanisms Contribute to Cold Tolerance in Plantain (Musa paradisiaca L.; ABB Group) Seedlings*

PubMed Central

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 proteomic analysis. PMID:22982374

Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC-MS/MS

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

Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC-MS/MS.

PubMed

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.

Proteomic analysis of the Theileria annulata schizont

PubMed Central

Witschi, M.; Xia, D.; Sanderson, S.; Baumgartner, M.; Wastling, J.M.; Dobbelaere, D.A.E.

2013-01-01

The apicomplexan parasite, Theileria annulata, is the causative agent of tropical theileriosis, a devastating lymphoproliferative disease of cattle. The schizont stage transforms bovine leukocytes and provides an intriguing model to study host/pathogen interactions. The genome of T. annulata has been sequenced and transcriptomic data are rapidly accumulating. In contrast, little is known about the proteome of the schizont, the pathogenic, transforming life cycle stage of the parasite. Using one-dimensional (1-D) gel LC-MS/MS, a proteomic analysis of purified T. annulata schizonts was carried out. In whole parasite lysates, 645 proteins were identified. Proteins with transmembrane domains (TMDs) were under-represented and no proteins with more than four TMDs could be detected. To tackle this problem, Triton X-114 treatment was applied, which facilitates the extraction of membrane proteins, followed by 1-D gel LC-MS/MS. This resulted in the identification of an additional 153 proteins. Half of those had one or more TMD and 30 proteins with more than four TMDs were identified. This demonstrates that Triton X-114 treatment can provide a valuable additional tool for the identification of new membrane proteins in proteomic studies. With two exceptions, all proteins involved in glycolysis and the citric acid cycle were identified. For at least 29% of identified proteins, the corresponding transcripts were not present in the existing expressed sequence tag databases. The proteomics data were integrated into the publicly accessible database resource at EuPathDB (www.eupathdb.org) so that mass spectrometry-based protein expression evidence for T. annulata can be queried alongside transcriptional and other genomics data available for these parasites. PMID:23178997

Human body fluid proteome analysis

PubMed Central

Hu, Shen; Loo, Joseph A.; Wong, David T.

2010-01-01

The focus of this article is to review the recent advances in proteome analysis of human body fluids, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, and amniotic fluid, as well as its applications to human disease biomarker discovery. We aim to summarize the proteomics technologies currently used for global identification and quantification of body fluid proteins, and elaborate the putative biomarkers discovered for a variety of human diseases through human body fluid proteome (HBFP) analysis. Some critical concerns and perspectives in this emerging field are also discussed. With the advances made in proteomics technologies, the impact of HBFP analysis in the search for clinically relevant disease biomarkers would be realized in the future. PMID:17083142

Human body fluid proteome analysis.

PubMed

Hu, Shen; Loo, Joseph A; Wong, David T

2006-12-01

The focus of this article is to review the recent advances in proteome analysis of human body fluids, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, and amniotic fluid, as well as its applications to human disease biomarker discovery. We aim to summarize the proteomics technologies currently used for global identification and quantification of body fluid proteins, and elaborate the putative biomarkers discovered for a variety of human diseases through human body fluid proteome (HBFP) analysis. Some critical concerns and perspectives in this emerging field are also discussed. With the advances made in proteomics technologies, the impact of HBFP analysis in the search for clinically relevant disease biomarkers would be realized in the future.

Micro-proteomics with iterative data analysis: Proteome analysis in C. elegans at the single worm level.

PubMed

Bensaddek, Dalila; Narayan, Vikram; Nicolas, Armel; Murillo, Alejandro Brenes; Gartner, Anton; Kenyon, Cynthia J; Lamond, Angus I

2016-02-01

Proteomics studies typically analyze proteins at a population level, using extracts prepared from tens of thousands to millions of cells. The resulting measurements correspond to average values across the cell population and can mask considerable variation in protein expression and function between individual cells or organisms. Here, we report the development of micro-proteomics for the analysis of Caenorhabditis elegans, a eukaryote composed of 959 somatic cells and ∼1500 germ cells, measuring the worm proteome at a single organism level to a depth of ∼3000 proteins. This includes detection of proteins across a wide dynamic range of expression levels (>6 orders of magnitude), including many chromatin-associated factors involved in chromosome structure and gene regulation. We apply the micro-proteomics workflow to measure the global proteome response to heat-shock in individual nematodes. This shows variation between individual animals in the magnitude of proteome response following heat-shock, including variable induction of heat-shock proteins. The micro-proteomics pipeline thus facilitates the investigation of stochastic variation in protein expression between individuals within an isogenic population of C. elegans. All data described in this study are available online via the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd), an open access, searchable database resource. © 2015 The Authors. PROTEOMICS Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

MASPECTRAS: a platform for management and analysis of proteomics LC-MS/MS data

PubMed Central

Hartler, Jürgen; Thallinger, Gerhard G; Stocker, Gernot; Sturn, Alexander; Burkard, Thomas R; Körner, Erik; Rader, Robert; Schmidt, Andreas; Mechtler, Karl; Trajanoski, Zlatko

2007-01-01

Background The advancements of proteomics technologies have led to a rapid increase in the number, size and rate at which datasets are generated. Managing and extracting valuable information from such datasets requires the use of data management platforms and computational approaches. Results We have developed the MAss SPECTRometry Analysis System (MASPECTRAS), a platform for management and analysis of proteomics LC-MS/MS data. MASPECTRAS is based on the Proteome Experimental Data Repository (PEDRo) relational database schema and follows the guidelines of the Proteomics Standards Initiative (PSI). Analysis modules include: 1) import and parsing of the results from the search engines SEQUEST, Mascot, Spectrum Mill, X! Tandem, and OMSSA; 2) peptide validation, 3) clustering of proteins based on Markov Clustering and multiple alignments; and 4) quantification using the Automated Statistical Analysis of Protein Abundance Ratios algorithm (ASAPRatio). The system provides customizable data retrieval and visualization tools, as well as export to PRoteomics IDEntifications public repository (PRIDE). MASPECTRAS is freely available at Conclusion Given the unique features and the flexibility due to the use of standard software technology, our platform represents significant advance and could be of great interest to the proteomics community. PMID:17567892

A simple protocol for protein extraction of recalcitrant fruit tissues suitable for 2-DE and MS analysis.

PubMed

Song, Jun; Braun, Gordon; Bevis, Eric; Doncaster, Kristen

2006-08-01

Fruit tissues are considered recalcitrant plant tissue for proteomic analysis. Three phenol-free protein extraction procedures for 2-DE were compared and evaluated on apple fruit proteins. Incorporation of hot SDS buffer, extraction with TCA/acetone precipitation was found to be the most effective protocol. The results from SDS-PAGE and 2-DE analysis showed high quality proteins. More than 500 apple polypeptides were separated on a small scale 2-DE gel. The successful protocol was further tested on banana fruit, in which 504 and 386 proteins were detected in peel and flesh tissues, respectively. To demonstrate the quality of the extracted proteins, several protein spots from apple and banana peels were cut from 2-DE gels, analyzed by MS and have been tentatively identified. The protocol described in this study is a simple procedure which could be routinely used in proteomic studies of many types of recalcitrant fruit tissues.

Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (Phospho)Proteomic Profiling.

PubMed

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.

Expanding proteome coverage with orthogonal-specificity α-Lytic proteases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyer, Jesse G.; Kim, Sangtae; Maltby, David A.

2014-03-01

Bottom-up proteomics studies traditionally involve proteome digestion with a single protease, trypsin. However, trypsin alone does not generate peptides that encompass the entire proteome. Alternative proteases have been explored, but most have specificity for charged amino acid side chains. Therefore, additional proteases that improve proteome coverage by cleavage at sequences complimentary to trypsin may increase proteome coverage. We demonstrate the novel application of two proteases for bottom-up proteomics: wild type alpha-lytic protease (WaLP), and an active site mutant of WaLP, M190A alpha-lytic protease (MaLP). We assess several relevant factors including MS/MS fragmentation, peptide length, peptide yield, and protease specificity. Bymore » combining data from separate digestions with trypsin, LysC, WaLP, and MaLP, proteome coverage was increased 101% compared to trypsin digestion alone. To demonstrate how the gained sequence coverage can access additional PTM information, we show identification of a number of novel phosphorylation sites in the S. pombe proteome and include an illustrative example from the protein MPD2, wherein two novel sites are identified, one in a tryptic peptide too short to identify and the other in a sequence devoid of tryptic sites. The specificity of WaLP and MaLP for aliphatic amino acid side chains was particularly valuable for coverage of membrane protein sequences, which increased 350% when the data from trypsin, LysC, WaLP, and MaLP were combined.« less

Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the pyochelin biosynthesis gene cluster.

PubMed

Wendler, Sergej; Otto, Andreas; Ortseifen, Vera; Bonn, Florian; Neshat, Armin; Schneiker-Bekel, Susanne; Walter, Frederik; Wolf, Timo; Zemke, Till; Wehmeier, Udo F; Hecker, Michael; Kalinowski, Jörn; Becher, Dörte; Pühler, Alfred

2015-07-01

Acarbose is an α-glucosidase inhibitor produced by Actinoplanes sp. SE50/110 that is medically important due to its application in the treatment of type2 diabetes. In this work, a comprehensive proteome analysis of Actinoplanes sp. SE50/110 was carried out to determine the location of proteins of the acarbose (acb) and the putative pyochelin (pch) biosynthesis gene cluster. Therefore, a comprehensive state-of-the-art proteomics approach combining subcellular fractionation, shotgun proteomics and spectral counting to assess the relative abundance of proteins within fractions was applied. The analysis of four different proteome fractions (cytosolic, enriched membrane, membrane shaving and extracellular fraction) resulted in the identification of 1582 of the 8270 predicted proteins. All 22 Acb-proteins and 21 of the 23 Pch-proteins were detected. Predicted membrane-associated, integral membrane or extracellular proteins of the pch and the acb gene cluster were found among the most abundant proteins in corresponding fractions. Intracellular biosynthetic proteins of both gene clusters were not only detected in the cytosolic, but also in the enriched membrane fraction, indicating that the biosynthesis of acarbose and putative pyochelin metabolites takes place at the inner membrane. Actinoplanes sp. SE50/110 is a natural producer of the α-glucosidase inhibitor acarbose, a bacterial secondary metabolite that is used as a drug for the treatment of type 2 diabetes, a disease which is a global pandemic that currently affects 387 million people and accounts for 11% of worldwide healthcare expenditures (www.idf.org). The work presented here is the first comprehensive investigation of protein localization and abundance in Actinoplanes sp. SE50/110 and provides an extensive source of information for the selection of genes for future mutational analysis and other hypothesis driven experiments. The conclusion that acarbose or pyochelin family siderophores are synthesized at the inner side of the cytoplasmic membrane determined from this work, indicates that studying corresponding intermediates will be challenging. In addition to previous studies on the genome and transcriptome, the work presented here demonstrates that the next omic level, the proteome, is now accessible for detailed physiological analysis of Actinoplanes sp. SE50/110, as well as mutants derived from this and related species. Copyright © 2015 Elsevier B.V. All rights reserved.

Integrated Proteomic Approaches for Understanding Toxicity of Environmental Chemicals

EPA Science Inventory

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...

Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties

PubMed Central

Zanni, Elena; Schifano, Emily; Motta, Sara; Sciubba, Fabio; Palleschi, Claudio; Mauri, Pierluigi; Perozzi, Giuditta; Uccelletti, Daniela; Devirgiliis, Chiara; Miccheli, Alfredo

2017-01-01

Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus, lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans, with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis. Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates. PMID:28702021

Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties.

PubMed

Zanni, Elena; Schifano, Emily; Motta, Sara; Sciubba, Fabio; Palleschi, Claudio; Mauri, Pierluigi; Perozzi, Giuditta; Uccelletti, Daniela; Devirgiliis, Chiara; Miccheli, Alfredo

2017-01-01

Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus , lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans , with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis . Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates.

Quantitative Phospho-proteomic Analysis of TNFα/NFκB Signaling Reveals a Role for RIPK1 Phosphorylation in Suppressing Necrotic Cell Death.

PubMed

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.

Quantitative proteomics reveals the mechanism and consequence of gliotoxin-mediated dysregulation of the methionine cycle in Aspergillus niger.

PubMed

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.

A Proteomic Analysis of Eccrine Sweat: Implications for the Discovery of Schizophrenia Biomarker Proteins

PubMed Central

Raiszadeh, Michelle M.; Ross, Mark M.; Russo, Paul S.; Schaepper, Mary Ann H.; Zhou, Weidong; Deng, Jianghong; Ng, Daniel; Dickson, April; Dickson, Cindy; Strom, Monica; Osorio, Carolina; Soeprono, Thomas; Wulfkuhle, Julia D.; Kabbani, Nadine; Petricoin, Emanuel F.; Liotta, Lance A.; Kirsch, Wolff M.

2012-01-01

Liquid chromatography tandem mass spectrometry (LC-MS/MS) and multiple reaction monitoring mass spectrometry (MRM-MS) proteomics analyses were performed on eccrine sweat of healthy controls, and the results were compared with those from individuals diagnosed with schizophrenia (SZ). This is the first large scale study of the sweat proteome. First, we performed LC-MS/MS on pooled SZ samples and pooled control samples for global proteomics analysis. Results revealed a high abundance of diverse proteins and peptides in eccrine sweat. Most of the proteins identified from sweat samples were found to be different than the most abundant proteins from serum, which indicates that eccrine sweat is not simply a plasma transudate, and may thereby be a source of unique disease-associated biomolecules. A second independent set of patient and control sweat samples were analyzed by LC-MS/MS and spectral counting to determine qualitative protein differential abundances between the control and disease groups. Differential abundances of selected proteins, initially determined by spectral counting, were verified by MRM-MS analyses. Seventeen proteins showed a differential abundance of approximately two-fold or greater between the SZ pooled sample and the control pooled sample. This study demonstrates the utility of LC-MS/MS and MRM-MS as a viable strategy for the discovery and verification of potential sweat protein disease biomarkers. PMID:22256890

Proteomic analysis of a decellularized human vocal fold mucosa scaffold using 2D electrophoresis and high-resolution mass spectrometry.

PubMed

Welham, Nathan V; Chang, Zhen; Smith, Lloyd M; Frey, Brian L

2013-01-01

Natural biologic scaffolds for tissue engineering are commonly generated by decellularization of tissues and organs. Despite some preclinical and clinical success, in vivo scaffold remodeling and functional outcomes remain variable, presumably due to the influence of unidentified bioactive molecules on the scaffold-host interaction. Here, we used 2D electrophoresis and high-resolution mass spectrometry-based proteomic analyses to evaluate decellularization effectiveness and identify potentially bioactive protein remnants in a human vocal fold mucosa model. We noted proteome, phosphoproteome and O-glycoproteome depletion post-decellularization, and identified >200 unique protein species within the decellularized scaffold. Gene ontology-based enrichment analysis revealed a dominant set of functionally-related ontology terms associated with extracellular matrix assembly, organization, morphology and patterning, consistent with preservation of a tissue-specific niche for later cell seeding and infiltration. We further identified a subset of ontology terms associated with bioactive (some of which are antigenic) cellular proteins, despite histological and immunohistochemical data indicating complete decellularization. These findings demonstrate the value of mass spectrometry-based proteomics in identifying agents potentially responsible for variation in host response to engineered tissues derived from decellularized scaffolds. This work has implications for the manufacturing of biologic scaffolds from any tissue or organ, as well as for prediction and monitoring of the scaffold-host interaction in vivo. Copyright © 2012 Elsevier Ltd. All rights reserved.

Proteomic approaches in research of cyanobacterial photosynthesis.

PubMed

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.

Oocyte spindle proteomics analysis leading to rescue of chromosome congression defects in cloned embryos

PubMed Central

Duan, Xunbao; Zhong, Zhisheng; Potireddy, Santhi; Moncada, Camilo; Merali, Salim; Latham, Keith E.

2015-01-01

Embryos produced by somatic cell nuclear transfer (SCNT) display low term developmental potential. This is associated with deficiencies in spindle composition prior to activation and at early mitotic divisions, including failure to assemble certain proteins on the spindle. The protein-deficient spindles are accompanied by chromosome congression defects prior to activation and during the first mitotic divisions of the embryo. The molecular basis for these deficiencies and how they might be avoided are unknown. Proteomic analyses of spindles isolated from normal metaphase II (MII) stage oocytes and SCNT constructs, along with a systematic immunofluorescent survey of known spindle-associated proteins were undertaken. This was the first proteomics study of mammalian oocyte spindles. The study revealed four proteins as being deficient in spindles of SCNT embryos in addition to those previously identified; these were clathrin heavy chain (CLTC), aurora B kinase, dynactin 4, and casein kinase 1 alpha. Due to substantial reduction in CLTC abundance after spindle removal, we undertook functional studies to explore the importance of CLTC in oocyte spindle function and in chromosome congression defects of cloned embryos. Using siRNA knockdown we demonstrated an essential role for CLTC in chromosome congression during oocyte maturation. We also demonstrated rescue of chromosome congression defects in SCNT embryos at the first mitosis using CLTC mRNA injection. These studies are the first to employ proteomics analyses coupled to functional interventions to rescue a specific molecular defect in cloned embryos. PMID:20883044

Unlocking Proteomic Heterogeneity in Complex Diseases through Visual Analytics

PubMed Central

Bhavnani, Suresh K.; Dang, Bryant; Bellala, Gowtham; Divekar, Rohit; Visweswaran, Shyam; Brasier, Allan; Kurosky, Alex

2015-01-01

Despite years of preclinical development, biological interventions designed to treat complex diseases like asthma often fail in phase III clinical trials. These failures suggest that current methods to analyze biomedical data might be missing critical aspects of biological complexity such as the assumption that cases and controls come from homogeneous distributions. Here we discuss why and how methods from the rapidly evolving field of visual analytics can help translational teams (consisting of biologists, clinicians, and bioinformaticians) to address the challenge of modeling and inferring heterogeneity in the proteomic and phenotypic profiles of patients with complex diseases. Because a primary goal of visual analytics is to amplify the cognitive capacities of humans for detecting patterns in complex data, we begin with an overview of the cognitive foundations for the field of visual analytics. Next, we organize the primary ways in which a specific form of visual analytics called networks have been used to model and infer biological mechanisms, which help to identify the properties of networks that are particularly useful for the discovery and analysis of proteomic heterogeneity in complex diseases. We describe one such approach called subject-protein networks, and demonstrate its application on two proteomic datasets. This demonstration provides insights to help translational teams overcome theoretical, practical, and pedagogical hurdles for the widespread use of subject-protein networks for analyzing molecular heterogeneities, with the translational goal of designing biomarker-based clinical trials, and accelerating the development of personalized approaches to medicine. PMID:25684269

CPTAC Releases Cancer Proteome Confirmatory Colon Study Data | Office of Cancer Clinical Proteomics Research

Cancer.gov

The National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) announces the release of the cancer proteome confirmatory colon study data. The goal of the study is to analyze the proteomes of approximately 100 confirmatory colon tumor patients, which includes tumor and adjacent normal samples, with liquid chromatography-tandem mass spectrometry (LC-MS/MS) global proteomic and phosphoproteomic profiling.

Myoferlin is a novel exosomal protein and functional regulator of cancer-derived exosomes.

PubMed

Blomme, Arnaud; Fahmy, Karim; Peulen, Olivier; Costanza, Brunella; Fontaine, Marie; Struman, Ingrid; Baiwir, Dominique; de Pauw, Edwin; Thiry, Marc; Bellahcène, Akeila; Castronovo, Vincent; Turtoi, Andrei

2016-12-13

Exosomes are communication mediators participating in the intercellular exchange of proteins, metabolites and nucleic acids. Recent studies have demonstrated that exosomes are characterized by a unique proteomic composition that is distinct from the cellular one. The mechanisms responsible for determining the proteome content of the exosomes remain however obscure. In the current study we employ ultrastructural approach to validate a novel exosomal protein myoferlin. This is a multiple C2-domain containing protein, known for its conserved physiological function in endocytosis and vesicle fusion biology. Emerging studies demonstrate that myoferlin is frequently overexpressed in cancer, where it promotes cancer cell migration and invasion. Our data expand these findings by showing that myoferlin is a general component of cancer cell derived exosomes from different breast and pancreatic cancer cell lines. Using proteomic analysis, we demonstrate for the first time that myoferlin depletion in cancer cells leads to a significantly modulated exosomal protein load. Such myoferlin-depleted exosomes were also functionally deficient as shown by their reduced capacity to transfer nucleic acids to human endothelial cells (HUVEC). Beyond this, myoferlin-depleted cancer exosomes also had a significantly reduced ability to induce migration and proliferation of HUVEC. The present study highlights myoferlin as a new functional player in exosome biology, calling for novel strategies to target this emerging oncogene in human cancer.

CPTAC Proteomics Data on UCSC Genome Browser | Office of Cancer Clinical Proteomics Research

Cancer.gov

The National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium scientists are working together with the University of California, Santa Cruz (UCSC) Genomics Institute to provide public access to cancer proteomics data via the UCSC Genome Browser. This effort extends accessibility of the CPTAC data to more researchers and provides an additional level of analysis to assist the cancer biology community.

Plant proteome analysis: a 2006 update.

PubMed

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 stresses, PTMs and Protein interactions. Section 8 summarizes the major pitfalls and challenges of plant proteomics.

CPTAC researchers report first large-scale integrated proteomic and genomic analysis of a human cancer | Office of Cancer Clinical Proteomics Research

Cancer.gov

Investigators from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) who comprehensively analyzed 95 human colorectal tumor samples, have determined how gene alterations identified in previous analyses of the same samples are expressed at the protein level. The integration of proteomic and genomic data, or proteogenomics, provides a more comprehensive view of the biological features that drive cancer than genomic analysis alone and may help identify the most important targets for cancer detection and intervention.

Analysis of Human Proteome Organization Plasma Proteome Project (HUPO PPP) reference specimens using surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry: multi-institution correlation of spectra and identification of biomarkers.

PubMed

Rai, Alex J; Stemmer, Paul M; Zhang, Zhen; Adam, Bao-Ling; Morgan, William T; Caffrey, Rebecca E; Podust, Vladimir N; Patel, Manisha; Lim, Lih-Yin; Shipulina, Natalia V; Chan, Daniel W; Semmes, O John; Leung, Hon-Chiu Eastwood

2005-08-01

We report on a multicenter analysis of HUPO reference specimens using SELDI-TOF MS. Eight sites submitted data obtained from serum and plasma reference specimen analysis. Spectra from five sites passed preliminary quality assurance tests and were subjected to further analysis. Intralaboratory CVs varied from 15 to 43%. A correlation coefficient matrix generated using data from these five sites demonstrated high level of correlation, with values >0.7 on 37 of 42 spectra. More than 50 peaks were differentially present among the various sample types, as observed on three chip surfaces. Additionally, peaks at approximately 9200 and approximately 15,950 m/z were present only in select reference specimens. Chromatographic fractionation using anion-exchange, membrane cutoff, and reverse phase chromatography, was employed for protein purification of the approximately 9200 m/z peak. It was identified as the haptoglobin alpha subunit after peptide mass fingerprinting and high-resolution MS/MS analysis. The differential expression of this protein was confirmed by Western blot analysis. These pilot studies demonstrate the potential of the SELDI platform for reproducible and consistent analysis of serum/plasma across multiple sites and also for targeted biomarker discovery and protein identification. This approach could be exploited for population-based studies in all phases of the HUPO PPP.

HIV-1 Vpr modulates macrophage metabolic pathways: a SILAC-based quantitative analysis.

PubMed

Barrero, Carlos A; Datta, Prasun K; Sen, Satarupa; Deshmane, Satish; Amini, Shohreh; Khalili, Kamel; Merali, Salim

2013-01-01

Human immunodeficiency virus type 1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Furthermore, these macrophages are resistant to cell death and are viral reservoir. However, the impact of Vpr on the macrophage proteome is yet to be comprehended. The goal of the present study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. Cultured human monocytic cells, U937, were differentiated into macrophages and transduced with adenovirus construct harboring the Vpr gene. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. Quantified proteins were selected and clustered by biological functions, pathway and network analysis using Ingenuity computational pathway analysis. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. In addition, the proteomic data demonstrate down regulation of some key mitochondrial enzymes such as glutamate dehydrogenase 2 (GLUD2), adenylate kinase 2 (AK2) and transketolase (TKT). Based on these observations we postulate that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehyrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. Furthermore, dysregulation of mitochondrial glutamate metabolism in macrophages can contribute to neurodegeneration via neuroexcitotoxic mechanisms in the context of NeuroAIDS.

Hormone regulation of rhizome development in tall fescue (Festuca arundinacea) associated with proteomic changes controlling respiratory and amino acid metabolism

PubMed Central

Ma, Xiqing; Xu, Qian; Meyer, William A.; Huang, Bingru

2016-01-01

Background and Aims Rhizomes are underground stems with meristematic tissues capable of generating shoots and roots. However, mechanisms controlling rhizome formation and growth are yet to be completely understood. The objectives of this study were to investigate whether rhizome development could be regulated by cytokinins (CKs) and gibberellic acids (GAs), and determine underlying mechanisms of regulation of rhizome formation and growth of tall fescue (Festuca arundinacea) by a CK or GA through proteomic and transcript analysis. Methods A rhizomatous genotype of tall fescue (‘BR’) plants were treated with 6-benzylaminopurine (BAP, a synthetic cytokinin) or GA3 in hydroponic culture in growth chambers. Furthermore, comparative proteomic analysis of two-dimensional electrophoresis and mass spectrometry were performed to investigate proteins and associated metabolic pathways imparting increased rhizome number by BAP and rhizome elongation by GA3. Key Results BAP stimulated rhizome formation while GA3 promoted rhizome elongation. Proteomic analysis identified 76 differentially expressed proteins (DEPs) due to BAP treatment and 37 DEPs due to GA3 treatment. Cytokinin-related genes and cell division-related genes were upregulated in the rhizome node by BAP and gibberellin-related and cell growth-related genes in the rhizome by GA3. Conclusions Most of the BAP- or GA-responsive DEPs were involved in respiratory metabolism and amino acid metabolism. Transcription analysis demonstrated that genes involved in hormone metabolism, signalling pathways, cell division and cell-wall loosening were upregulated by BAP or GA3. The CK and GA promoted rhizome formation and growth, respectively, by activating metabolic pathways that supply energy and amino acids to support cell division and expansion during rhizome initiation and elongation in tall fescue. PMID:27443301

A Proteomics View of the Molecular Mechanisms and Biomarkers of Glaucomatous Neurodegeneration

PubMed Central

Tezel, Gülgün

2013-01-01

Despite improving understanding of glaucoma, key molecular players of neurodegeneration that can be targeted for treatment of glaucoma, or molecular biomarkers that can be useful for clinical testing, remain unclear. Proteomics technology offers a powerful toolbox to accomplish these important goals of the glaucoma research and is increasingly being applied to identify molecular mechanisms and biomarkers of glaucoma. Recent studies of glaucoma using proteomics analysis techniques have resulted in the lists of differentially expressed proteins in human glaucoma and animal models. The global analysis of protein expression in glaucoma has been followed by cell-specific proteome analysis of retinal ganglion cells and astrocytes. The proteomics data have also guided targeted studies to identify post-translational modifications and protein-protein interactions during glaucomatous neurodegeneration. In addition, recent applications of proteomics have provided a number of potential biomarker candidates. Proteomics technology holds great promise to move glaucoma research forward toward new treatment strategies and biomarker discovery. By reviewing the major proteomics approaches and their applications in the field of glaucoma, this article highlights the power of proteomics in translational and clinical research related to glaucoma and also provides a framework for future research to functionally test the importance of specific molecular pathways and validate candidate biomarkers. PMID:23396249

Comparative proteomics lends insight into genotype-specific pathogenicity.

PubMed

Guarnieri, Michael T

2013-09-01

Comparative proteomic analyses have emerged as a powerful tool for the identification of unique biomarkers and mechanisms of pathogenesis. In this issue of Proteomics, Murugaiyan et al. utilize difference gel electrophoresis (DIGE) to examine differential protein expression between nonpathogenic and pathogenic genotypes of Prototheca zopfii, a causative agent in bovine enteritis and mastitis. Their findings provide insights into molecular mechanisms of infection and evolutionary adaptation of pathogenic genotypes, demonstrating the power of comparative proteomic analyses. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Jing; Ma, Zihao; Carr, Steven A.

Coexpression of mRNAs under multiple conditions is commonly used to infer cofunctionality of their gene products despite well-known limitations of this “guilt-by-association” (GBA) approach. Recent advancements in mass spectrometry-based proteomic technologies have enabled global expression profiling at the protein level; however, whether proteome profiling data can outperform transcriptome profiling data for coexpression based gene function prediction has not been systematically investigated. Here, we address this question by constructing and analyzing mRNA and protein coexpression networks for three cancer types with matched mRNA and protein profiling data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC).more » Our analyses revealed a marked difference in wiring between the mRNA and protein coexpression networks. Whereas protein coexpression was driven primarily by functional similarity between coexpressed genes, mRNA coexpression was driven by both cofunction and chromosomal colocalization of the genes. Functionally coherent mRNA modules were more likely to have their edges preserved in corresponding protein networks than functionally incoherent mRNA modules. Proteomic data strengthened the link between gene expression and function for at least 75% of Gene Ontology (GO) biological processes and 90% of KEGG pathways. A web application Gene2Net (http://cptac.gene2net.org) developed based on the three protein coexpression networks revealed novel gene-function relationships, such as linking ERBB2 (HER2) to lipid biosynthetic process in breast cancer, identifying PLG as a new gene involved in complement activation, and identifying AEBP1 as a new epithelial-mesenchymal transition (EMT) marker. Our results demonstrate that proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction. Proteomics should be integrated if not preferred in gene function and human disease studies. Molecular & Cellular Proteomics 16: 10.1074/mcp.M116.060301, 121–134, 2017.« less

Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients.

PubMed

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.

Proteomic characterization of EL4 lymphoma-derived tumors upon chemotherapy treatment reveals potential roles for lysosomes and caspase-6 during tumor cell death in vivo.

PubMed

Kramer, David A; Eldeeb, Mohamed A; Wuest, Melinda; Mercer, John; Fahlman, Richard P

2017-06-01

The murine mouse lymphoblastic lymphoma cell line (EL4) tumor model is an established in vivo apoptosis model for the investigation of novel cancer imaging agents and immunological treatments due to the rapid and significant response of the EL4 tumors to cyclophosphamide and etoposide combination chemotherapy. Despite the utility of this model system in cancer research, little is known regarding the molecular details of in vivo tumor cell death. Here, we report the first in-depth quantitative proteomic analysis of the changes that occur in these tumors upon cyclophosphamide and etoposide treatment in vivo. Using a label-free quantitative proteomic approach a total of 5838 proteins were identified in the treated and untreated tumors, of which 875 were determined to change in abundance with statistical significance. Initial analysis of the data reveals changes that may have been predicted, such as the downregulation of ribosomes, but demonstrates the robustness of the dataset. Analysis of the dataset also reveals the unexpected downregulation of caspase-3 and an upregulation of caspase-6 in addition to a global upregulation of lysosomal proteins in the bulk of the tumor. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DeMix Workflow for Efficient Identification of Cofragmented Peptides in High Resolution Data-dependent Tandem Mass Spectrometry*

PubMed Central

Zhang, Bo; Pirmoradian, Mohammad; Chernobrovkin, Alexey; Zubarev, Roman A.

2014-01-01

Based on conventional data-dependent acquisition strategy of shotgun proteomics, we present a new workflow DeMix, which significantly increases the efficiency of peptide identification for in-depth shotgun analysis of complex proteomes. Capitalizing on the high resolution and mass accuracy of Orbitrap-based tandem mass spectrometry, we developed a simple deconvolution method of “cloning” chimeric tandem spectra for cofragmented peptides. Additional to a database search, a simple rescoring scheme utilizes mass accuracy and converts the unwanted cofragmenting events into a surprising advantage of multiplexing. With the combination of cloning and rescoring, we obtained on average nine peptide-spectrum matches per second on a Q-Exactive workbench, whereas the actual MS/MS acquisition rate was close to seven spectra per second. This efficiency boost to 1.24 identified peptides per MS/MS spectrum enabled analysis of over 5000 human proteins in single-dimensional LC-MS/MS shotgun experiments with an only two-hour gradient. These findings suggest a change in the dominant “one MS/MS spectrum - one peptide” paradigm for data acquisition and analysis in shotgun data-dependent proteomics. DeMix also demonstrated higher robustness than conventional approaches in terms of lower variation among the results of consecutive LC-MS/MS runs. PMID:25100859

[Search for potential gastric cancer biomarkers using low molecular weight blood plasma proteome profiling by mass spectrometry].

PubMed

Shevchenko, V E; Arnotskaia, N E; Ogorodnikova, E V; Davydov, M M; Ibraev, M A; Turkin, I N; Davydov, M I

2014-01-01

Gastric cancer, one of the most widespread malignant tumors, still lacks reliable serum/plasma biomarkers of its early detection. In this study we have developed, unified, and tested a new methodology for search of gastric cancer biomarkers based on profiling of low molecular weight proteome (LMWP) (1-17 kDa). This approach included three main components: sample pre-fractionation, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), data analysis by a bioinformatics software package. Applicability and perspectives of the developed approach for detection of potential gastric cancer markers during LMWP analysis have been demonstrated using 69 plasma samples from patients with gastric cancer (stages I-IV) and 238 control samples. The study revealed peptides/polypeptides, which may be potentially used for detection of this pathology.

Proteomic-based identification of Apg-2 as a therapeutic target for chronic myeloid leukemia.

PubMed

Li, Yajuan; Chen, Xi; Shi, Meng; Wang, Haixia; Cao, Weixi; Wang, Xiaozhong; Li, Chunli; Feng, Wenli

2013-12-01

The oncogenic BCR/ABL tyrosine kinase induces constitutive enhanced "spontaneous" DNA damage and unfaithful repair in Philadelphia chromosome positive leukemia cells. Here, we investigated the changes of protein profile in H2O2-induced DNA damage/repair in BaF3-MIGR1 and BaF3-BCR/ABL cells through a proteomic strategy consisting of two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF mass spectrometry. In total, 41 spots were differentially expressed and 13 proteins were identified with further MS analysis. Two essential proteins, Proto-oncogene tyrosine-protein kinase ABL1 (c-ABL) and Heat shock 70kDa protein 4 (Apg-2), were confirmed by Western blot and showed consistent changes with proteomic results. Moreover, functional analysis demonstrated that inhibition of Apg-2 not only decreased cell proliferation, but also induced cell apoptosis in BCR/ABL positive cells (BaF3-BCR/ABL, BaF3-BCR/ABL(T315I)). We also proved that Apg-2 inhibition aggravated H2O2 induced damage in BCR/ABL positive cells, and enhanced the sensitivity of BaF3-BCR/ABL(T315I) to STI571. Taken together, the findings in this work provide us with some clues to a better understanding of the molecular mechanisms underlying BCR/ABL in the DNA damage/repair processes and demonstrated that Apg-2 would be a valid target for anti-leukemia drug development. © 2013.

FunRich proteomics software analysis, let the fun begin!

PubMed

Benito-Martin, Alberto; Peinado, Héctor

2015-08-01

Protein MS analysis is the preferred method for unbiased protein identification. It is normally applied to a large number of both small-scale and high-throughput studies. However, user-friendly computational tools for protein analysis are still needed. In this issue, Mathivanan and colleagues (Proteomics 2015, 15, 2597-2601) report the development of FunRich software, an open-access software that facilitates the analysis of proteomics data, providing tools for functional enrichment and interaction network analysis of genes and proteins. FunRich is a reinterpretation of proteomic software, a standalone tool combining ease of use with customizable databases, free access, and graphical representations. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative Proteomics Analysis of Streptomyces coelicolor Development Demonstrates That Onset of Secondary Metabolism Coincides with Hypha Differentiation*

PubMed Central

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

Top-down proteomics for the analysis of proteolytic events - Methods, applications and perspectives.

PubMed

Tholey, Andreas; Becker, Alexander

2017-11-01

Mass spectrometry based proteomics is an indispensable tool for almost all research areas relevant for the understanding of proteolytic processing, ranging from the identification of substrates, products and cleavage sites up to the analysis of structural features influencing protease activity. The majority of methods for these studies are based on bottom-up proteomics performing analysis at peptide level. As this approach is characterized by a number of pitfalls, e.g. loss of molecular information, there is an ongoing effort to establish top-down proteomics, performing separation and MS analysis both at intact protein level. We briefly introduce major approaches of bottom-up proteomics used in the field of protease research and highlight the shortcomings of these methods. We then discuss the present state-of-the-art of top-down proteomics. Together with the discussion of known challenges we show the potential of this approach and present a number of successful applications of top-down proteomics in protease research. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John. Copyright © 2017 Elsevier B.V. All rights reserved.

Shotgun proteomics of plant plasma membrane and microdomain proteins using nano-LC-MS/MS.

PubMed

Takahashi, Daisuke; Li, Bin; Nakayama, Takato; Kawamura, Yukio; Uemura, Matsuo

2014-01-01

Shotgun proteomics allows the comprehensive analysis of proteins extracted from plant cells, subcellular organelles, and membranes. Previously, two-dimensional gel electrophoresis-based proteomics was used for mass spectrometric analysis of plasma membrane proteins. In order to get comprehensive proteome profiles of the plasma membrane including highly hydrophobic proteins with a number of transmembrane domains, a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for proteins from the plasma membrane proteins and plasma membrane microdomain fraction is described. The results obtained are easily applicable to label-free protein semiquantification.

Proteomic and metabolomic analysis on the toxicological effects of As (III) and As (V) in juvenile mussel Mytilus galloprovincialis.

PubMed

Yu, Deliang; Ji, Chenglong; Zhao, Jianmin; Wu, Huifeng

2016-05-01

Inorganic arsenic (As) is a known pollutant including two chemical forms (arsenite (As III) and arsenate (As V)), in marine and coastal environment. Marine mussel Mytilus galloprovincialis is an important environmental monitoring species around the world. In this study, we focused on valence-specific responses of As in juvenile mussel M. galloprovincialis using a combined proteomic and metabolomic approach. Metabolic responses indicated that As (III) mainly caused disturbance in osmotic regulation in juvenile mussels. As (V) caused disturbances in both osmotic regulation and energy metabolism marked by different metabolic responses, including betaine, taurine, glucose and glycogen. Proteomic responses exhibited that As (III) had a significant negative effect on cytoskeleton and cell structure (actin and collagen alpha-6(VI) chain). As (V) affected some key enzymes involved in energy metabolism (cytosolic malate dehydrogenase, cMDH) and cell development (ornithine aminotransferase and astacin). Overall, all these results confirmed the valence-specific responses in juvenile mussels to As exposures. These findings demonstrate that a combined metabolomic and proteomic approach could provide an important insight into the toxicological effects of environmental pollutants in organisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

A tutorial in displaying mass spectrometry-based proteomic data using heat maps.

PubMed

Key, Melissa

2012-01-01

Data visualization plays a critical role in interpreting experimental results of proteomic experiments. Heat maps are particularly useful for this task, as they allow us to find quantitative patterns across proteins and biological samples simultaneously. The quality of a heat map can be vastly improved by understanding the options available to display and organize the data in the heat map. This tutorial illustrates how to optimize heat maps for proteomics data by incorporating known characteristics of the data into the image. First, the concepts used to guide the creating of heat maps are demonstrated. Then, these concepts are applied to two types of analysis: visualizing spectral features across biological samples, and presenting the results of tests of statistical significance. For all examples we provide details of computer code in the open-source statistical programming language R, which can be used for biologists and clinicians with little statistical background. Heat maps are a useful tool for presenting quantitative proteomic data organized in a matrix format. Understanding and optimizing the parameters used to create the heat map can vastly improve both the appearance and the interoperation of heat map data.

Development of an Efficient Virus Induced Gene Silencing Strategy in the Non-Model Wild Ginger-Zingiber zerumbet and Investigation of Associated Proteome Changes

PubMed Central

Mahadevan, Chidambareswaren; Jaleel, Abdul; Deb, Lokesh; Thomas, George; Sakuntala, Manjula

2015-01-01

Zingiber zerumbet (Zingiberaceae) is a wild, tropical medicinal herb that shows a high degree of resistance to diseases affecting cultivated ginger. Barley stripe mosaic virus (BSMV) silencing vectors containing an endogenous phytoene desaturase (PDS) gene fragment were agroinfiltrated into young leaves of Z. zerumbet under controlled growth conditions to effect virus-induced gene silencing (VIGS). Infiltrated leaves as well as newly emerged leaves and tillers showed visual signs of PDS silencing after 30 days. Replication and systemic movement of the viral vectors in silenced plants were confirmed by RT-PCR. Real-time quantitative PCR analysis verified significant down-regulation of PDS transcripts in the silenced tissues. Label-free proteomic analysis was conducted in leaves with established PDS transcript down regulation and buffer-infiltrated (mock) leaves. A total of 474 proteins were obtained, which were up-regulated, down-regulated or modulated de novo during VIGS. Most of these proteins were localized to the chloroplast, as revealed by UniprotKB analysis, and among the up-regulated proteins there were abiotic stress responsive, photosynthetic, metabolic and membrane proteins. Moreover, the demonstration of viral proteins together with host proteins proved successful viral infection. We report for the first time the establishment of a high-throughput gene functional analysis platform using BSMV-mediated VIGS in Z. zerumbet, as well as proteomic changes associated with VIGS. PMID:25918840

Evaluation of selected binding domains for the analysis of ubiquitinated proteomes

PubMed Central

Nakayasu, Ernesto S.; Ansong, Charles; Brown, Joseph N.; Yang, Feng; Lopez-Ferrer, Daniel; Qian, Wei-Jun; Smith, Richard D.; Adkins, Joshua N.

2013-01-01

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising, but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ∼200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle. PMID:23649778

Combined Proteomic and Transcriptomic Interrogation of the Venom Gland of Conus geographus Uncovers Novel Components and Functional Compartmentalization*

PubMed Central

Safavi-Hemami, Helena; Hu, Hao; Gorasia, Dhana G.; Bandyopadhyay, Pradip K.; Veith, Paul D.; Young, Neil D.; Reynolds, Eric C.; Yandell, Mark; Olivera, Baldomero M.; Purcell, Anthony W.

2014-01-01

Cone snails are highly successful marine predators that use complex venoms to capture prey. At any given time, hundreds of toxins (conotoxins) are synthesized in the secretory epithelial cells of the venom gland, a long and convoluted organ that can measure 4 times the length of the snail's body. In recent years a number of studies have begun to unveil the transcriptomic, proteomic and peptidomic complexity of the venom and venom glands of a number of cone snail species. By using a combination of DIGE, bottom-up proteomics and next-generation transcriptome sequencing the present study identifies proteins involved in envenomation and conotoxin maturation, significantly extending the repertoire of known (poly)peptides expressed in the venom gland of these remarkable animals. We interrogate the molecular and proteomic composition of different sections of the venom glands of 3 specimens of the fish hunter Conus geographus and demonstrate regional variations in gene expression and protein abundance. DIGE analysis identified 1204 gel spots of which 157 showed significant regional differences in abundance as determined by biological variation analysis. Proteomic interrogation identified 342 unique proteins including those that exhibited greatest fold change. The majority of these proteins also exhibited significant changes in their mRNA expression levels validating the reliability of the experimental approach. Transcriptome sequencing further revealed a yet unknown genetic diversity of several venom gland components. Interestingly, abundant proteins that potentially form part of the injected venom mixture, such as echotoxins, phospholipase A2 and con-ikots-ikots, classified into distinct expression clusters with expression peaking in different parts of the gland. Our findings significantly enhance the known repertoire of venom gland polypeptides and provide molecular and biochemical evidence for the compartmentalization of this organ into distinct functional entities. PMID:24478445

Epsilon-Q: An Automated Analyzer Interface for Mass Spectral Library Search and Label-Free Protein Quantification.

PubMed

Cho, Jin-Young; Lee, Hyoung-Joo; Jeong, Seul-Ki; Paik, Young-Ki

2017-12-01

Mass spectrometry (MS) is a widely used proteome analysis tool for biomedical science. In an MS-based bottom-up proteomic approach to protein identification, sequence database (DB) searching has been routinely used because of its simplicity and convenience. However, searching a sequence DB with multiple variable modification options can increase processing time, false-positive errors in large and complicated MS data sets. Spectral library searching is an alternative solution, avoiding the limitations of sequence DB searching and allowing the detection of more peptides with high sensitivity. Unfortunately, this technique has less proteome coverage, resulting in limitations in the detection of novel and whole peptide sequences in biological samples. To solve these problems, we previously developed the "Combo-Spec Search" method, which uses manually multiple references and simulated spectral library searching to analyze whole proteomes in a biological sample. In this study, we have developed a new analytical interface tool called "Epsilon-Q" to enhance the functions of both the Combo-Spec Search method and label-free protein quantification. Epsilon-Q performs automatically multiple spectral library searching, class-specific false-discovery rate control, and result integration. It has a user-friendly graphical interface and demonstrates good performance in identifying and quantifying proteins by supporting standard MS data formats and spectrum-to-spectrum matching powered by SpectraST. Furthermore, when the Epsilon-Q interface is combined with the Combo-Spec search method, called the Epsilon-Q system, it shows a synergistic function by outperforming other sequence DB search engines for identifying and quantifying low-abundance proteins in biological samples. The Epsilon-Q system can be a versatile tool for comparative proteome analysis based on multiple spectral libraries and label-free quantification.

Developmental distribution of the plasma membrane-enriched proteome in the maize primary root growth zone.

PubMed

Zhang, Zhe; Voothuluru, Priyamvada; Yamaguchi, Mineo; Sharp, Robert E; Peck, Scott C

2013-01-01

Within the growth zone of the maize primary root, there are well-defined patterns of spatial and temporal organization of cell division and elongation. However, the processes underlying this organization remain poorly understood. To gain additional insights into the differences amongst the defined regions, we performed a proteomic analysis focusing on fractions enriched for plasma membrane (PM) proteins. The PM is the interface between the plant cell and the apoplast and/or extracellular space. As such, it is a key structure involved in the exchange of nutrients and other molecules as well as in the integration of signals that regulate growth and development. Despite the important functions of PM-localized proteins in mediating these processes, a full understanding of dynamic changes in PM proteomes is often impeded by low relative concentrations relative to total proteins. Using a relatively simple strategy of treating microsomal fractions with Brij-58 detergent to enrich for PM proteins, we compared the developmental distribution of proteins within the root growth zone which revealed a number of previously known as well as novel proteins with interesting patterns of abundance. For instance, the quantitative proteomic analysis detected a gradient of PM aquaporin proteins similar to that previously reported using immunoblot analyses, confirming the veracity of this strategy. Cellulose synthases increased in abundance with increasing distance from the root apex, consistent with expected locations of cell wall deposition. The similar distribution pattern for Brittle-stalk-2-like protein implicates that this protein may also have cell wall related functions. These results show that the simplified PM enrichment method previously demonstrated in Arabidopsis can be successfully applied to completely unrelated plant tissues and provide insights into differences in the PM proteome throughout growth and development zones of the maize primary root.

Proteomic Changes in Rat Thyroarytenoid Muscle Induced by Botulinum Neurotoxin Injection

PubMed Central

Welham, Nathan V.; Marriott, Gerard; Tateya, Ichiro; Bless, Diane M.

2009-01-01

Botulinum neurotoxin (BoNT) injection into the thyroarytenoid (TA) muscle is a commonly performed medical intervention for adductor spasmodic dysphonia. The mechanism of action of BoNT at the neuromuscular junction is well understood, however, aside from reports focused on myosin heavy chain isoform abundance, there is a paucity of data addressing the effects of therapeutic BoNT injection on the TA muscle proteome. In this study, 12 adult Sprague Dawley rats underwent unilateral TA muscle BoNT serotype A injection followed by tissue harvest at 72 hrs, 7 days, 14 days, and 56 days post-injection. Three additional rats were reserved as controls. Proteomic analysis was performed using 2D SDS-PAGE followed by MALDI-MS. Vocal fold movement was significantly reduced by 72 hrs, with complete return of function by 56 days. Twenty-five protein spots demonstrated significant protein abundance changes following BoNT injection, and were associated with alterations in energy metabolism, muscle contractile function, cellular stress response, transcription, translation, and cell proliferation. A number of protein abundance changes persisted beyond the return of gross physiologic TA function. These findings represent the first report of BoNT induced changes in any skeletal muscle proteome, and reinforce the utility of applying proteomic tools to the study of system-wide biological processes in normal and perturbed TA muscle function. PMID:18442174

Learning from Heterogeneous Data Sources: An Application in Spatial Proteomics

PubMed Central

Breckels, Lisa M.; Holden, Sean B.; Wojnar, David; Mulvey, Claire M.; Christoforou, Andy; Groen, Arnoud; Trotter, Matthew W. B.; Kohlbacher, Oliver; Lilley, Kathryn S.; Gatto, Laurent

2016-01-01

Sub-cellular localisation of proteins is an essential post-translational regulatory mechanism that can be assayed using high-throughput mass spectrometry (MS). These MS-based spatial proteomics experiments enable us to pinpoint the sub-cellular distribution of thousands of proteins in a specific system under controlled conditions. Recent advances in high-throughput MS methods have yielded a plethora of experimental spatial proteomics data for the cell biology community. Yet, there are many third-party data sources, such as immunofluorescence microscopy or protein annotations and sequences, which represent a rich and vast source of complementary information. We present a unique transfer learning classification framework that utilises a nearest-neighbour or support vector machine system, to integrate heterogeneous data sources to considerably improve on the quantity and quality of sub-cellular protein assignment. We demonstrate the utility of our algorithms through evaluation of five experimental datasets, from four different species in conjunction with four different auxiliary data sources to classify proteins to tens of sub-cellular compartments with high generalisation accuracy. We further apply the method to an experiment on pluripotent mouse embryonic stem cells to classify a set of previously unknown proteins, and validate our findings against a recent high resolution map of the mouse stem cell proteome. The methodology is distributed as part of the open-source Bioconductor pRoloc suite for spatial proteomics data analysis. PMID:27175778

Proteomic analysis of middle and late stages of bread wheat (Triticum aestivum L.) grain development

PubMed Central

Zhang, Ning; Chen, Feng; Huo, Wang; Cui, Dangqun

2015-01-01

Proteomic approaches were applied in four grain developmental stages of the Chinese bread wheat Yunong 201 and its ethyl methanesulfonate (EMS) mutant line Yunong 3114. 2-DE and tandem MALDI-TOF/TOF-MS analyzed proteome characteristics during middle and late grain development of the Chinese bread wheat Yunong 201 and its EMS mutant line Yunong 3114 with larger grain sizes. We identified 130 differentially accumulated protein spots representing 88 unique proteins, and four main expression patterns displayed a dynamic description of middle and late grain formation. Those identified protein species participated in eight biochemical processes: stress/defense, carbohydrate metabolism, protein synthesis/assembly/degradation, storage proteins, energy production and transportation, photosynthesis, transcription/translation, signal transduction. Comparative proteomic characterization demonstrated 12 protein spots that co-accumulated in the two wheat cultivars with different expression patterns, and six cultivar-specific protein spots including serpin, small heat shock protein, β-amylase, α-amylase inhibitor, dimeric α-amylase inhibitor precursor, and cold regulated protein. These cultivar-specific protein spots possibly resulted in differential yield-related traits of the two wheat cultivars. Our results provide valuable information for dissection of molecular and genetics basis of yield-related traits in bread wheat and the proteomic characterization in this study could also provide insights in the biology of middle and late grain development. PMID:26442048

Analysis of Intrinsic Peptide Detectability via Integrated Label-Free and SRM-Based Absolute Quantitative Proteomics.

PubMed

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.

Mathematical biodescriptors of proteomics maps: background and applications.

PubMed

Basak, Subhash C; Gute, Brian D

2008-05-01

This article reviews recent developments in the formulation and application of biodescriptors to characterize proteomics maps. Such biodescriptors can be derived by applying techniques from discrete mathematics (graph theory, linear algebra and information theory). This review focuses on the development of biodescriptors for proteomics maps derived from 2D gel electrophoresis. Preliminary results demonstrated that such descriptors have a reasonable ability to differentiate between proteomics patterns that result from exposure to closely related individual chemicals and complex mixtures, such as the jet fuel JP-8. Further research is required to evaluate the utility of these proteomics-based biodescriptors for drug discovery and predictive toxicology.

Experimental design and data-analysis in label-free quantitative LC/MS proteomics: A tutorial with MSqRob.

PubMed

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.

ProteoSign: an end-user online differential proteomics statistical analysis platform.

PubMed

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.

The Escherichia coli Proteome: Past, Present, and Future Prospects†

PubMed Central

Han, Mee-Jung; Lee, Sang Yup

2006-01-01

Proteomics has emerged as an indispensable methodology for large-scale protein analysis in functional genomics. The Escherichia coli proteome has been extensively studied and is well defined in terms of biochemical, biological, and biotechnological data. Even before the entire E. coli proteome was fully elucidated, the largest available data set had been integrated to decipher regulatory circuits and metabolic pathways, providing valuable insights into global cellular physiology and the development of metabolic and cellular engineering strategies. With the recent advent of advanced proteomic technologies, the E. coli proteome has been used for the validation of new technologies and methodologies such as sample prefractionation, protein enrichment, two-dimensional gel electrophoresis, protein detection, mass spectrometry (MS), combinatorial assays with n-dimensional chromatographies and MS, and image analysis software. These important technologies will not only provide a great amount of additional information on the E. coli proteome but also synergistically contribute to other proteomic studies. Here, we review the past development and current status of E. coli proteome research in terms of its biological, biotechnological, and methodological significance and suggest future prospects. PMID:16760308

Tumor Cold Ischemia | Office of Cancer Clinical Proteomics Research

Cancer.gov

In a recently published manuscript in the journal of Molecular and Cellular Proteomics, researchers from the National Cancer Institutes (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) investigated the effect of cold ischemia on the proteome of fresh frozen tumors.

Proteomics wants cRacker: automated standardized data analysis of LC-MS derived proteomic data.

PubMed

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.

Building ProteomeTools based on a complete synthetic human proteome

PubMed Central

Zolg, Daniel P.; Wilhelm, Mathias; Schnatbaum, Karsten; Zerweck, Johannes; Knaute, Tobias; Delanghe, Bernard; Bailey, Derek J.; Gessulat, Siegfried; Ehrlich, Hans-Christian; Weininger, Maximilian; Yu, Peng; Schlegl, Judith; Kramer, Karl; Schmidt, Tobias; Kusebauch, Ulrike; Deutsch, Eric W.; Aebersold, Ruedi; Moritz, Robert L.; Wenschuh, Holger; Moehring, Thomas; Aiche, Stephan; Huhmer, Andreas; Reimer, Ulf; Kuster, Bernhard

2018-01-01

The ProteomeTools project builds molecular and digital tools from the human proteome to facilitate biomedical and life science research. Here, we report the generation and multimodal LC-MS/MS analysis of >330,000 synthetic tryptic peptides representing essentially all canonical human gene products and exemplify the utility of this data. The resource will be extended to >1 million peptides and all data will be shared with the community via ProteomicsDB and proteomeXchange. PMID:28135259

Characterization of individual mouse cerebrospinal fluid proteomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Jeffrey S.; Angel, Thomas E.; Chavkin, Charles

2014-03-20

Analysis of cerebrospinal fluid (CSF) offers key insight into the status of the central nervous system. Characterization of murine CSF proteomes can provide a valuable resource for studying central nervous system injury and disease in animal models. However, the small volume of CSF in mice has thus far limited individual mouse proteome characterization. Through non-terminal CSF extractions in C57Bl/6 mice and high-resolution liquid chromatography-mass spectrometry analysis of individual murine samples, we report the most comprehensive proteome characterization of individual murine CSF to date. Utilizing stringent protein inclusion criteria that required the identification of at least two unique peptides (1% falsemore » discovery rate at the peptide level) we identified a total of 566 unique proteins, including 128 proteins from three individual CSF samples that have been previously identified in brain tissue. Our methods and analysis provide a mechanism for individual murine CSF proteome analysis.« less

Deep coverage of the beer proteome.

PubMed

Grochalová, Martina; Konečná, Hana; Stejskal, Karel; Potěšil, David; Fridrichová, Danuše; Srbová, Eva; Ornerová, Kateřina; Zdráhal, Zbyněk

2017-06-06

We adopted an approach based on peptide immobilized pH gradient-isoelectric focusing (IPG-IEF) separation, coupled with LC-MS/MS, in order to maximize coverage of the beer proteome. A lager beer brewed using traditional Czech technology was degassed, desalted and digested. Tryptic peptides were separated by isoelectric focusing on an immobilized pH gradient strip and, after separation, the gel strip was divided into seven equally sized parts. Peptides extracted from gel fractions were analyzed by LC-MS/MS. This approach resulted in a three-fold increase in the number of proteins identified (over 1700) when compared to analysis of unfractionated beer processed by a filter-aided sample preparation (FASP). Over 1900 protein groups (PGs) in total were identified by both approaches. The study significantly extends knowledge about the beer proteome and demonstrates its complexity. Detailed knowledge of the protein content, especially gluten proteins, will enhance the evaluation of potential health risks related to beer consumption (coeliac disease) and will contribute to improving beer quality. Copyright © 2017 Elsevier B.V. All rights reserved.

Improvement of Quantitative Measurements in Multiplex Proteomics Using High-Field Asymmetric Waveform Spectrometry.

PubMed

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.

Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues.

PubMed

Ku, Taeyun; Swaney, Justin; Park, Jeong-Yoon; Albanese, Alexandre; Murray, Evan; Cho, Jae Hun; Park, Young-Gyun; Mangena, Vamsi; Chen, Jiapei; Chung, Kwanghun

2016-09-01

The biology of multicellular organisms is coordinated across multiple size scales, from the subnanoscale of molecules to the macroscale, tissue-wide interconnectivity of cell populations. Here we introduce a method for super-resolution imaging of the multiscale organization of intact tissues. The method, called magnified analysis of the proteome (MAP), linearly expands entire organs fourfold while preserving their overall architecture and three-dimensional proteome organization. MAP is based on the observation that preventing crosslinking within and between endogenous proteins during hydrogel-tissue hybridization allows for natural expansion upon protein denaturation and dissociation. The expanded tissue preserves its protein content, its fine subcellular details, and its organ-scale intercellular connectivity. We use off-the-shelf antibodies for multiple rounds of immunolabeling and imaging of a tissue's magnified proteome, and our experiments demonstrate a success rate of 82% (100/122 antibodies tested). We show that specimen size can be reversibly modulated to image both inter-regional connections and fine synaptic architectures in the mouse brain.

Examination of Triacylglycerol Biosynthetic Pathways via De Novo Transcriptomic and Proteomic Analyses in an Unsequenced Microalga

PubMed Central

Guarnieri, Michael T.; Nag, Ambarish; Smolinski, Sharon L.; Darzins, Al; Seibert, Michael; Pienkos, Philip T.

2011-01-01

Biofuels derived from algal lipids represent an opportunity to dramatically impact the global energy demand for transportation fuels. Systems biology analyses of oleaginous algae could greatly accelerate the commercialization of algal-derived biofuels by elucidating the key components involved in lipid productivity and leading to the initiation of hypothesis-driven strain-improvement strategies. However, higher-level systems biology analyses, such as transcriptomics and proteomics, are highly dependent upon available genomic sequence data, and the lack of these data has hindered the pursuit of such analyses for many oleaginous microalgae. In order to examine the triacylglycerol biosynthetic pathway in the unsequenced oleaginous microalga, Chlorella vulgaris, we have established a strategy with which to bypass the necessity for genomic sequence information by using the transcriptome as a guide. Our results indicate an upregulation of both fatty acid and triacylglycerol biosynthetic machinery under oil-accumulating conditions, and demonstrate the utility of a de novo assembled transcriptome as a search model for proteomic analysis of an unsequenced microalga. PMID:22043295

[Methods of quantitative proteomics].

PubMed

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.

Magnetic capture of polydopamine-encapsulated Hela cells for the analysis of cell surface proteins.

PubMed

Liu, Yiying; Yan, Guoquan; Gao, Mingxia; Zhang, Xiangmin

2018-02-10

A novel method to characterize cell surface proteins and complexes has been developed. Polydopamine (PDA)-encapsulated Hela cells were prepared for plasma membrane proteome research. Since the PDA protection, the encapsulated cells could be maintained for more than two weeks. Amino groups functionalized magnetic nanoparticles were also used for cell capture by the reaction with the PDA coatings. Plasma membrane fragments were isolated and enriched with assistance of an external magnetic field after disruption of the coated cells by ultrasonic treatment. Plasma membrane proteins (PMPs) and complexes were well preserved on the fragments and identified by shot-gun proteomic analytical strategy. 385 PMPs and 1411 non-PMPs were identified using the method. 85.2% of these PMPs were lipid-raft associated proteins. Ingenuity Pathway Analysis was employed for bio-information extraction from the identified proteins. It was found that 653 non-PMPs had interactions with 140 PMPs. Among them, epidermal growth factor receptor and its complexes, and a series of important pathways including STAT3 pathway were observed. All these results demonstrated that the new approach is of great importance in applying to the research of physiological function and mechanism of the plasma membrane proteins. This work developed a novel strategy for the proteomic analysis of cell surface proteins. According to the results, 73.3% of total identified proteins were lipid-raft associated proteins, which imply that the proposed method is of great potential in the identification of lipid-raft associated proteins. In addition, a series of protein-protein interactions and pathways related to Hela cells were pointed out. All these results demonstrated that our proposed approach is of great importance and could well be applied to the physiological function and mechanism research of plasma membrane proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Saturated or unsaturated fat supplemented maternal diets influence omental adipose tissue proteome of suckling goat-kids.

PubMed

Restelli, Laura; Marques, Andreia T; Savoini, Giovanni; Invernizzi, Guido; Carisetti, Michela; Lecchi, Cristina; Bendixen, Emoke; Ceciliani, Fabrizio

2017-11-03

The aim of the present study was to investigate how maternal diet can influence the adipose tissue of goat kids. Omental adipose tissue proteomes of goat-kids from mothers fed with diet enriched with stearic acid (ST-kids), fish oil (FO-kids) and standard diets (CTRL) were determined by quantitative iTRAQ 2D-LC-MS/MS analysis. Twenty proteins were found to be differentially expressed in suckling kids' omental adipose tissue. Stearic acid induces changes in a higher number of proteins when compared to fish oil. Eleven proteins, namely AARS, ECl1, PMSC2, CP, HSPA8, GPD1, RPL7, OGDH, RPL24, FGA and RPL5 were decreased in ST-kids only. Four proteins, namely DLST, EEF1G, BCAP31 and RALA were decreased in FO-kids only, and one, NUCKS1, was increased. Four proteins, namely PMSC1, PPIB, TUB5×2 and EIF5A1, were be less abundant in both ST- and FO- kids. Most of the protein whose abundance was decreased in ST kids (10 out of 15) are involved in protein metabolism and catabolism pathways. Qualitative gene expression analysis confirmed that all the proteins identified by mass spectrometry, with the exception of FGA, were produced by adipose tissue. Quantitative gene expression analysis demonstrated that two proteins, namely CP, a minor acute phase protein, and ECl1, involved in fatty acid beta oxidation, were downregulated at mRNA level as well. ECl1 gene expression was downregulated in ST-kids AT as compared to Ctrl-kids and CP was downregulated in both ST- and FO-kids. The present results demonstrate that it is possible to influence adipose goat-kid proteome by modifying the maternal diet. Copyright © 2017. Published by Elsevier Ltd.

A novel algorithm for validating peptide identification from a shotgun proteomics search engine.

PubMed

Jian, Ling; Niu, Xinnan; Xia, Zhonghang; Samir, Parimal; Sumanasekera, Chiranthani; Mu, Zheng; Jennings, Jennifer L; Hoek, Kristen L; Allos, Tara; Howard, Leigh M; Edwards, Kathryn M; Weil, P Anthony; Link, Andrew J

2013-03-01

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has revolutionized the proteomics analysis of complexes, cells, and tissues. In a typical proteomic analysis, the tandem mass spectra from a LC-MS/MS experiment are assigned to a peptide by a search engine that compares the experimental MS/MS peptide data to theoretical peptide sequences in a protein database. The peptide spectra matches are then used to infer a list of identified proteins in the original sample. However, the search engines often fail to distinguish between correct and incorrect peptides assignments. In this study, we designed and implemented a novel algorithm called De-Noise to reduce the number of incorrect peptide matches and maximize the number of correct peptides at a fixed false discovery rate using a minimal number of scoring outputs from the SEQUEST search engine. The novel algorithm uses a three-step process: data cleaning, data refining through a SVM-based decision function, and a final data refining step based on proteolytic peptide patterns. Using proteomics data generated on different types of mass spectrometers, we optimized the De-Noise algorithm on the basis of the resolution and mass accuracy of the mass spectrometer employed in the LC-MS/MS experiment. Our results demonstrate De-Noise improves peptide identification compared to other methods used to process the peptide sequence matches assigned by SEQUEST. Because De-Noise uses a limited number of scoring attributes, it can be easily implemented with other search engines.

Comprehensive Analysis of Temporal Alterations in Cellular Proteome of Bacillus subtilis under Curcumin Treatment

PubMed Central

Reddy, Panga Jaipal; Sinha, Sneha; Ray, Sandipan; Sathe, Gajanan J.; Chatterjee, Aditi; Prasad, T. S. Keshava; Dhali, Snigdha; Srikanth, Rapole; Panda, Dulal; Srivastava, Sanjeeva

2015-01-01

Curcumin is a natural dietary compound with antimicrobial activity against various gram positive and negative bacteria. This study aims to investigate the proteome level alterations in Bacillus subtilis due to curcumin treatment and identification of its molecular/cellular targets to understand the mechanism of action. We have performed a comprehensive proteomic analysis of B. subtilis AH75 strain at different time intervals of curcumin treatment (20, 60 and 120 min after the drug exposure, three replicates) to compare the protein expression profiles using two complementary quantitative proteomic techniques, 2D-DIGE and iTRAQ. To the best of our knowledge, this is the first comprehensive longitudinal investigation describing the effect of curcumin treatment on B. subtilis proteome. The proteomics analysis revealed several interesting targets such UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1, putative septation protein SpoVG and ATP-dependent Clp protease proteolytic subunit. Further, in silico pathway analysis using DAVID and KOBAS has revealed modulation of pathways related to the fatty acid metabolism and cell wall synthesis, which are crucial for cell viability. Our findings revealed that curcumin treatment lead to inhibition of the cell wall and fatty acid synthesis in addition to differential expression of many crucial proteins involved in modulation of bacterial metabolism. Findings obtained from proteomics analysis were further validated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) assay for respiratory activity, resazurin assay for metabolic activity and membrane integrity assay by potassium and inorganic phosphate leakage measurement. The gene expression analysis of selected cell wall biosynthesis enzymes has strengthened the proteomics findings and indicated the major effect of curcumin on cell division. PMID:25874956

Comprehensive analysis of temporal alterations in cellular proteome of Bacillus subtilis under curcumin treatment.

PubMed

Reddy, Panga Jaipal; Sinha, Sneha; Ray, Sandipan; Sathe, Gajanan J; Chatterjee, Aditi; Prasad, T S Keshava; Dhali, Snigdha; Srikanth, Rapole; Panda, Dulal; Srivastava, Sanjeeva

2015-01-01

Curcumin is a natural dietary compound with antimicrobial activity against various gram positive and negative bacteria. This study aims to investigate the proteome level alterations in Bacillus subtilis due to curcumin treatment and identification of its molecular/cellular targets to understand the mechanism of action. We have performed a comprehensive proteomic analysis of B. subtilis AH75 strain at different time intervals of curcumin treatment (20, 60 and 120 min after the drug exposure, three replicates) to compare the protein expression profiles using two complementary quantitative proteomic techniques, 2D-DIGE and iTRAQ. To the best of our knowledge, this is the first comprehensive longitudinal investigation describing the effect of curcumin treatment on B. subtilis proteome. The proteomics analysis revealed several interesting targets such UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1, putative septation protein SpoVG and ATP-dependent Clp protease proteolytic subunit. Further, in silico pathway analysis using DAVID and KOBAS has revealed modulation of pathways related to the fatty acid metabolism and cell wall synthesis, which are crucial for cell viability. Our findings revealed that curcumin treatment lead to inhibition of the cell wall and fatty acid synthesis in addition to differential expression of many crucial proteins involved in modulation of bacterial metabolism. Findings obtained from proteomics analysis were further validated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) assay for respiratory activity, resazurin assay for metabolic activity and membrane integrity assay by potassium and inorganic phosphate leakage measurement. The gene expression analysis of selected cell wall biosynthesis enzymes has strengthened the proteomics findings and indicated the major effect of curcumin on cell division.

Discovery of potential protein biomarkers of lung adenocarcinoma in bronchoalveolar lavage fluid by SWATH MS data-independent acquisition and targeted data extraction.

PubMed

Ortea, I; Rodríguez-Ariza, A; Chicano-Gálvez, E; Arenas Vacas, M S; Jurado Gámez, B

2016-04-14

Lung cancer currently ranks as the neoplasia with the highest global mortality rate. Although some improvements have been introduced in recent years, new advances in diagnosis are required in order to increase survival rates. New mildly invasive endoscopy-based diagnostic techniques include the collection of bronchoalveolar lavage fluid (BALF), which is discarded after using a portion of the fluid for standard pathological procedures. BALF proteomic analysis can contribute to clinical practice with more sensitive biomarkers, and can complement cytohistological studies by aiding in the diagnosis, prognosis, and subtyping of lung cancer, as well as the monitoring of treatment response. The range of quantitative proteomics methodologies used for biomarker discovery is currently being broadened with the introduction of data-independent acquisition (DIA) analysis-related approaches that address the massive quantitation of the components of a proteome. Here we report for the first time a DIA-based quantitative proteomics study using BALF as the source for the discovery of potential lung cancer biomarkers. The results have been encouraging in terms of the number of identified and quantified proteins. A panel of candidate protein biomarkers for adenocarcinoma in BALF is reported; this points to the activation of the complement network as being strongly over-represented and suggests this pathway as a potential target for lung cancer research. In addition, the results reported for haptoglobin, complement C4-A, and glutathione S-transferase pi are consistent with previous studies, which indicates that these proteins deserve further consideration as potential lung cancer biomarkers in BALF. Our study demonstrates that the analysis of BALF proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS), combining a simple sample pre-treatment and SWATH DIA MS, is a useful method for the discovery of potential lung cancer biomarkers. Bronchoalveolar lavage fluid (BALF) analysis can contribute to clinical practice with more sensitive biomarkers, thus complementing cytohistological studies in order to aid in the diagnosis, prognosis, and subtyping of lung cancer, as well as the monitoring of treatment response. Here we report a panel of candidate protein biomarkers for adenocarcinoma in BALF. Forty-four proteins showed a fold-change higher than 3.75 among adenocarcinoma patients compared with controls. This report is the first DIA-based quantitative proteomics study to use bronchoalveolar lavage fluid (BALF) as a matrix for discovering potential biomarkers. The results are encouraging in terms of the number of identified and quantified proteins, demonstrating that the analysis of BALF proteins by a SWATH approach is a useful method for the discovery of potential biomarkers of pulmonary diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Unparalleled sample treatment throughput for proteomics workflows relying on ultrasonic energy.

PubMed

Jorge, Susana; Araújo, J E; Pimentel-Santos, F M; Branco, Jaime C; Santos, Hugo M; Lodeiro, Carlos; Capelo, J L

2018-02-01

We report on the new microplate horn ultrasonic device as a powerful tool to speed proteomics workflows with unparalleled throughput. 96 complex proteomes were digested at the same time in 4min. Variables such as ultrasonication time, ultrasonication amplitude, and protein to enzyme ratio were optimized. The "classic" method relying on overnight protein digestion (12h) and the sonoreactor-based method were also employed for comparative purposes. We found the protein digestion efficiency homogeneously distributed in the entire microplate horn surface using the following conditions: 4min sonication time and 25% amplitude. Using this approach, patients with lymphoma and myeloma were classified using principal component analysis and a 2D gel-mass spectrometry based approach. Furthermore, we demonstrate the excellent performance by using MALDI-mass spectrometry based profiling as a fast way to classify patients with rheumatoid arthritis, systemic lupus erythematosus, and ankylosing spondylitis. Finally, the speed and simplicity of this method were demonstrated by clustering 90 patients with knee osteoarthritis disease (30), with a prosthesis (30, control group) and healthy individuals (30) with no history of joint disease. Overall, the new approach allows profiling a disease in just one week while allows to match the minimalism rules as outlined by Halls. Copyright © 2017 Elsevier B.V. All rights reserved.

Proteomic Signatures of Acquired Letrozole Resistance in Breast Cancer: Suppressed Estrogen Signaling and Increased Cell Motility and Invasiveness*

PubMed Central

Tilghman, Syreeta L.; Townley, Ian; Zhong, Qiu; Carriere, Patrick P.; Zou, Jin; Llopis, Shawn D.; Preyan, Lynez C.; Williams, Christopher C.; Skripnikova, Elena; Bratton, Melyssa R.; Zhang, Qiang; Wang, Guangdi

2013-01-01

Aromatase inhibitors, such as letrozole, have become the first-line treatment for postmenopausal women with estrogen-dependent breast cancer. However, acquired resistance remains a major clinical obstacle. Previous studies demonstrated constitutive activation of the MAPK signaling, overexpression of HER2, and down-regulation of aromatase and ERα in letrozole-resistant breast cancer cells. Given the complex signaling network involved in letrozole-refractory breast cancer and the lack of effective treatment for hormone resistance, further investigation of aromatase inhibitor resistance by a novel systems biology approach may reveal previously unconsidered molecular changes that could be utilized as therapeutic targets. This study was undertaken to characterize for the first time global proteomic alterations occurring in a letrozole-resistant cell line. A quantitative proteomic analysis of the whole cell lysates of LTLT-Ca (resistant) versus AC-1 cells (sensitive) was performed to identify significant protein expression changes. A total of 1743 proteins were identified and quantified, of which 411 were significantly up-regulated and 452 significantly down-regulated (p < 0.05, fold change > 1.20). Bioinformatics analysis revealed that acquired letrozole resistance is associated with a hormone-independent, more aggressive phenotype. LTLT-Ca cells exhibited 84% and 138% increase in migration and invasion compared with the control cells. The ROCK inhibitor partially abrogated the enhanced migration and invasion of the letrozole-resistant cells. Flow cytometric analyses also demonstrated an increase in vimentin and twist expression in letrozole-resistance cells, suggesting an onset of epithelial to mesenchymal transition (EMT). Moreover, targeted gene expression arrays confirmed a 28-fold and sixfold up-regulation of EGFR and HER2, respectively, whereas ERα and pS2 were dramatically reduced by 28-fold and 1100-fold, respectively. Taken together, our study revealed global proteomic signatures of a letrozole-resistant cell line associated with hormone independence, enhanced cell motility, EMT and the potential values of several altered proteins as novel prognostic markers or therapeutic targets for letrozole resistant breast cancer. PMID:23704778

Analyzing large-scale proteomics projects with latent semantic indexing.

PubMed

Klie, Sebastian; Martens, Lennart; Vizcaíno, Juan Antonio; Côté, Richard; Jones, Phil; Apweiler, Rolf; Hinneburg, Alexander; Hermjakob, Henning

2008-01-01

Since the advent of public data repositories for proteomics data, readily accessible results from high-throughput experiments have been accumulating steadily. Several large-scale projects in particular have contributed substantially to the amount of identifications available to the community. Despite the considerable body of information amassed, very few successful analyses have been performed and published on this data, leveling off the ultimate value of these projects far below their potential. A prominent reason published proteomics data is seldom reanalyzed lies in the heterogeneous nature of the original sample collection and the subsequent data recording and processing. To illustrate that at least part of this heterogeneity can be compensated for, we here apply a latent semantic analysis to the data contributed by the Human Proteome Organization's Plasma Proteome Project (HUPO PPP). Interestingly, despite the broad spectrum of instruments and methodologies applied in the HUPO PPP, our analysis reveals several obvious patterns that can be used to formulate concrete recommendations for optimizing proteomics project planning as well as the choice of technologies used in future experiments. It is clear from these results that the analysis of large bodies of publicly available proteomics data by noise-tolerant algorithms such as the latent semantic analysis holds great promise and is currently underexploited.

Evolution of Clinical Proteomics and its Role in Medicine | Office of Cancer Clinical Proteomics Research

Cancer.gov

NCI's Office of Cancer Clinical Proteomics Research authored a review of the current state of clinical proteomics in the peer-reviewed Journal of Proteome Research. The review highlights outcomes from the CPTC program and also provides a thorough overview of the different technologies that have pushed the field forward. Additionally, the review provides a vision for moving the field forward through linking advances in genomic and proteomic analysis to develop new, molecularly targeted interventions.

ApoptoProteomics, an integrated database for analysis of proteomics data obtained from apoptotic cells.

PubMed

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.

ApoptoProteomics, an Integrated Database for Analysis of Proteomics Data Obtained from Apoptotic Cells*

PubMed Central

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

Clinical proteomic analysis of scrub typhus infection.

PubMed

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.

TRIC: an automated alignment strategy for reproducible protein quantification in targeted proteomics.

PubMed

Röst, Hannes L; Liu, Yansheng; D'Agostino, Giuseppe; Zanella, Matteo; Navarro, Pedro; Rosenberger, George; Collins, Ben C; Gillet, Ludovic; Testa, Giuseppe; Malmström, Lars; Aebersold, Ruedi

2016-09-01

Next-generation mass spectrometric (MS) techniques such as SWATH-MS have substantially increased the throughput and reproducibility of proteomic analysis, but ensuring consistent quantification of thousands of peptide analytes across multiple liquid chromatography-tandem MS (LC-MS/MS) runs remains a challenging and laborious manual process. To produce highly consistent and quantitatively accurate proteomics data matrices in an automated fashion, we developed TRIC (http://proteomics.ethz.ch/tric/), a software tool that utilizes fragment-ion data to perform cross-run alignment, consistent peak-picking and quantification for high-throughput targeted proteomics. TRIC reduced the identification error compared to a state-of-the-art SWATH-MS analysis without alignment by more than threefold at constant recall while correcting for highly nonlinear chromatographic effects. On a pulsed-SILAC experiment performed on human induced pluripotent stem cells, TRIC was able to automatically align and quantify thousands of light and heavy isotopic peak groups. Thus, TRIC fills a gap in the pipeline for automated analysis of massively parallel targeted proteomics data sets.

Chemical-Biological Properties of Zinc Sensors TSQ and Zinquin: Formation of Sensor-Zn-Protein Adducts versus Zn(Sensor)2 Complexes.

PubMed

Nowakowski, Andrew B; Meeusen, Jeffrey W; Menden, Heather; Tomasiewicz, Henry; Petering, David H

2015-12-21

Fluorescent zinc sensors are the most commonly used tool to study the intracellular mobile zinc status within cellular systems. Previously, we have shown that the quinoline-based sensors Zinquin and 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ) predominantly form ternary adducts with members of the Zn-proteome. Here, the chemistries of these sensors are further characterized, including how Zn(sensor)2 complexes may react in an intracellular environment. We demonstrate that these sensors are typically used in higher concentrations than needed to obtain maximum signal. Exposing cells to either Zn(Zinquin)2 or Zn(TSQ)2 resulted in efficient cellular uptake and the formation of sensor-Zn-protein adducts as evidenced by both a fluorescence spectral shift toward that of ternary adducts and the localization of the fluorescence signal within the proteome after gel filtration of cellular lysates. Likewise, reacting Zn(sensor)2 with the Zn-proteome from LLC-PK1 cells resulted in the formation of sensor-Zn-protein ternary adducts that could be inhibited by first saturating the Zn- proteome with excess sensor. Further, a native SDS-PAGE analysis of the Zn-proteome reacted with either the sensor or the Zn(sensor)2 complex revealed that both reactions result in the formation of a similar set of sensor-Zn-protein fluorescent products. The results of this experiment also demonstrated that TSQ and Zinquin react with different members of the Zn-proteome. Reactions with the model apo-Zn-protein bovine serum albumin showed that both Zn(TSQ)2 and Zn(Zinquin)2 reacted to form ternary adducts with its apo-Zn-binding site. Moreover, incubating Zn(sensor)2 complexes with non-zinc binding proteins failed to elicit a spectral shift in the fluorescence spectrum, supporting the premise that blue-shifted emission spectra are due to sensor-Zn-protein ternary adducts. It was concluded that Zn(sensors)2 species do not play a significant role in the overall reaction between these sensors and intact cells. In turn, this study further supports the formation of sensor-Zn-protein adducts as the principal observed fluorescent product during experiments employing these two sensors.

Saliva analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) in orthodontic treatment: first pilot study.

PubMed

Ciavarella, Domenico; Mastrovincenzo, Mario; D'Onofrio, Valentina; Chimenti, Claudio; Parziale, Vincenzo; Barbato, Ersilia; Lo Muzio, Lorenzo

2011-11-01

SELDI-TOF-MS (Surface-Enhanced Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) allows the generation of an accurate protein profile from minimal amounts of biological samples and may executes proteomic profile of saliva. The aim of this work is to compare the proteomic profile of saliva of patients in orthodontic treatment to the beginning of treatment and after three months by using the surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology. Saliva was collected from 14 patients, between the 11 and 17 years, to the beginning of the orthodontic treatment and after three months. Specimens were centrifuged (10 min, 13000 x g); the Q10 ProteinChips were prepared according to the manufacturer's instructions and were loaded with the supernatants. A saturated solution of sinapinic acid was used as energy-absorbing matrix. The analysis was performed in a m/z range from 2500 to 25000 Da, and the proteomic profiles were compared by a specific data analysis software. Saliva (5 mL) was collected by spitting directly into a clean 15 mL conical tube. The samples were then aliquotted and stored at -80°C until use. Profile of saliva of patients before orthodontic treatment present a number of peaks different respect profile of saliva after three months of treatment. The average intensities of peaks at m/z 3372, 5232, 4045 and 10128 were significantly higher after three months then at beginning of treatment in the same patients and among these one. The Roc Plot has demonstrated high sensitivity and specificity. Many differences were noted in salivary proteomic profile obtained using the SELDI-TOF-MS technology in patients in orthodontic treatment to beginning and after three months. These data suggest that the proteomic analysis of saliva is a promising new tool for a non-invasive study of oral mucosa and bone changes. Copyright © 2011 Società Italiana di Ortodonzia SIDO. Published by Elsevier Srl. All rights reserved.

Targeted Approach for Proteomic Analysis of a Hidden Membrane Protein.

PubMed

Martins-Marques, Tania; Anjo, Sandra I; Ribeiro-Rodrigues, Teresa; Manadas, Bruno; Girao, Henrique

2017-01-01

Given the properties of plasma membrane proteins, namely, their hydrophobicity, low solubility, and high resistance to digestion and extraction, their identification by traditional mass spectrometry (MS) has been a challenging task. Hence, proteomic studies involving the transmembrane protein connexin43 (Cx43) are scarce. Additionally, studies demonstrating the presence of proteins embedded in the lipid bilayer of extracellular vesicles (EVs) are difficult to perform and require specific changes and fine adjustments in the experimental and technical procedure to allow their detection by MS. In this review, we provide a detailed description of the protocol we have used to detect Cx43 in EVs of human peripheral blood. This includes some of the modifications that we have introduced in order to improve the detection of Cx43 in EVs, including an optimization of vesicle isolation, Cx43 purification, MS acquisition data, and further analysis.

Quantitative proteome analysis of barley seeds using ruthenium(II)-tris-(bathophenanthroline-disulphonate) staining.

PubMed

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 these limitations the application of a more sensitive stain would lead to a more advanced proteome approach. In summary, we demonstrate here that the application of RuBP as an economical but reliable and sensitive fluorescence stain is highly suitable for quantitative proteome analysis of plant seeds.

Marine proteomics: a critical assessment of an emerging technology.

PubMed

Slattery, Marc; Ankisetty, Sridevi; Corrales, Jone; Marsh-Hunkin, K Erica; Gochfeld, Deborah J; Willett, Kristine L; Rimoldi, John M

2012-10-26

The application of proteomics to marine sciences has increased in recent years because the proteome represents the interface between genotypic and phenotypic variability and, thus, corresponds to the broadest possible biomarker for eco-physiological responses and adaptations. Likewise, proteomics can provide important functional information regarding biosynthetic pathways, as well as insights into mechanism of action, of novel marine natural products. The goal of this review is to (1) explore the application of proteomics methodologies to marine systems, (2) assess the technical approaches that have been used, and (3) evaluate the pros and cons of this proteomic research, with the intent of providing a critical analysis of its future roles in marine sciences. To date, proteomics techniques have been utilized to investigate marine microbe, plant, invertebrate, and vertebrate physiology, developmental biology, seafood safety, susceptibility to disease, and responses to environmental change. However, marine proteomics studies often suffer from poor experimental design, sample processing/optimization difficulties, and data analysis/interpretation issues. Moreover, a major limitation is the lack of available annotated genomes and proteomes for most marine organisms, including several "model species". Even with these challenges in mind, there is no doubt that marine proteomics is a rapidly expanding and powerful integrative molecular research tool from which our knowledge of the marine environment, and the natural products from this resource, will be significantly expanded.

Preprocessing and Analysis of LC-MS-Based Proteomic Data

PubMed Central

Tsai, Tsung-Heng; Wang, Minkun; Ressom, Habtom W.

2016-01-01

Liquid chromatography coupled with mass spectrometry (LC-MS) has been widely used for profiling protein expression levels. This chapter is focused on LC-MS data preprocessing, which is a crucial step in the analysis of LC-MS based proteomics. We provide a high-level overview, highlight associated challenges, and present a step-by-step example for analysis of data from LC-MS based untargeted proteomic study. Furthermore, key procedures and relevant issues with the subsequent analysis by multiple reaction monitoring (MRM) are discussed. PMID:26519169

High-coverage quantitative proteomics using amine-specific isotopic labeling.

PubMed

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.

Global Proteome Analysis Links Lysine Acetylation to Diverse Functions in Oryza Sativa.

PubMed

Xue, Chao; Liu, Shuai; Chen, Chen; Zhu, Jun; Yang, Xibin; Zhou, Yong; Guo, Rui; Liu, Xiaoyu; Gong, Zhiyun

2018-01-01

Lysine acetylation (Kac) is an important protein post-translational modification in both eukaryotes and prokaryotes. Herein, we report the results of a global proteome analysis of Kac and its diverse functions in rice (Oryza sativa). We identified 1353 Kac sites in 866 proteins in rice seedlings. A total of 11 Kac motifs are conserved, and 45% of the identified proteins are localized to the chloroplast. Among all acetylated proteins, 38 Kac sites are combined in core histones. Bioinformatics analysis revealed that Kac occurs on a diverse range of proteins involved in a wide variety of biological processes, especially photosynthesis. Protein-protein interaction networks of the identified proteins provided further evidence that Kac contributes to a wide range of regulatory functions. Furthermore, we demonstrated that the acetylation level of histone H3 (lysine 27 and 36) is increased in response to cold stress. In summary, our approach comprehensively profiles the regulatory roles of Kac in the growth and development of rice. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of latexin by a proteomic analysis in rat normal articular cartilage

PubMed Central

2010-01-01

Background Osteoarthritis (OA) is characterized by degeneration of articular cartilage. Animal models of OA induced are a widely used tool in the study of the pathogenesis of disease. Several proteomic techniques for selective extraction of proteins have provided protein profiles of chondrocytes and secretory patterns in normal and osteoarthritic cartilage, including the discovery of new and promising biomarkers. In this proteomic analysis to study several proteins from rat normal articular cartilage, two-dimensional electrophoresis and mass spectrometry (MS) were used. Interestingly, latexin (LXN) was found. Using an immunohistochemical technique, it was possible to determine its localization within the chondrocytes from normal and osteoarthritic articular cartilage. Results In this study, 147 proteins were visualized, and 47 proteins were identified by MS. A significant proportion of proteins are involved in metabolic processes and energy (32%), as well as participating in different biological functions including structural organization (19%), signal transduction and molecular signaling (11%), redox homeostasis (9%), transcription and protein synthesis (6%), and transport (6%). The identified proteins were assigned to one or more subcellular compartments. Among the identified proteins, we found some proteins already recognized in other studies such as OA-associated proteins. Interestingly, we identified LXN, an inhibitor of mammalian carboxypeptidases, which had not been described in articular cartilage. Immunolabeling assays for LXN showed a granular distribution pattern in the cytoplasm of most chondrocytes of the middle, deep and calcified zones of normal articular cartilage as well as in subchondral bone. In osteoarthritic cartilage, LXN was observed in superficial and deep zones. Conclusions This study provides the first proteomic analysis of normal articular cartilage of rat. We identified LXN, whose location was demonstrated by immunolabeling in the chondrocytes from the middle, deep and calcified zones of normal articular cartilage, and superficial and deep zones of osteoarthritic cartilage. PMID:20525390

Bioinformatics analysis reveals biophysical and evolutionary insights into the 3-nitrotyrosine post-translational modification in the human proteome

PubMed Central

Ng, John Y.; Boelen, Lies; Wong, Jason W. H.

2013-01-01

Protein 3-nitrotyrosine is a post-translational modification that commonly arises from the nitration of tyrosine residues. This modification has been detected under a wide range of pathological conditions and has been shown to alter protein function. Whether 3-nitrotyrosine is important in normal cellular processes or is likely to affect specific biological pathways remains unclear. Using GPS-YNO2, a recently described 3-nitrotyrosine prediction algorithm, a set of predictions for nitrated residues in the human proteome was generated. In total, 9.27 per cent of the proteome was predicted to be nitratable (27 922/301 091). By matching the predictions against a set of curated and experimentally validated 3-nitrotyrosine sites in human proteins, it was found that GPS-YNO2 is able to predict 73.1 per cent (404/553) of these sites. Furthermore, of these sites, 42 have been shown to be nitrated endogenously, with 85.7 per cent (36/42) of these predicted to be nitrated. This demonstrates the feasibility of using the predicted dataset for a whole proteome analysis. A comprehensive bioinformatics analysis was subsequently performed on predicted and all experimentally validated nitrated tyrosine. This found mild but specific biophysical constraints that affect the susceptibility of tyrosine to nitration, and these may play a role in increasing the likelihood of 3-nitrotyrosine to affect processes, including phosphorylation and DNA binding. Furthermore, examining the evolutionary conservation of predicted 3-nitrotyrosine showed that, relative to non-nitrated tyrosine residues, 3-nitrotyrosine residues are generally less conserved. This suggests that, at least in the majority of cases, 3-nitrotyrosine is likely to have a deleterious effect on protein function and less likely to be important in normal cellular function. PMID:23389939

An integrated proteomic and transcriptomic analysis of perivitelline fluid proteins in a freshwater gastropod laying aerial eggs.

PubMed

Mu, Huawei; Sun, Jin; Heras, Horacio; Chu, Ka Hou; Qiu, Jian-Wen

2017-02-23

Proteins of the egg perivitelline fluid (PVF) that surrounds the embryo are critical for embryonic development in many animals, but little is known about their identities. Using an integrated proteomic and transcriptomic approach, we identified 64 proteins from the PVF of Pomacea maculata, a freshwater snail adopting aerial oviposition. Proteins were classified into eight functional groups: major multifunctional perivitellin subunits, immune response, energy metabolism, protein degradation, oxidation-reduction, signaling and binding, transcription and translation, and others. Comparison of gene expression levels between tissues showed that 22 PVF genes were exclusively expressed in albumen gland, the female organ that secretes PVF. Base substitution analysis of PVF and housekeeping genes between P. maculata and its closely related species Pomacea canaliculata showed that the reproductive proteins had a higher mean evolutionary rate. Predicted 3D structures of selected PVF proteins showed that some nonsynonymous substitutions are located at or near the binding regions that may affect protein function. The proteome and sequence divergence analysis revealed a substantial amount of maternal investment in embryonic nutrition and defense, and higher adaptive selective pressure on PVF protein-coding genes when compared with housekeeping genes, providing insight into the adaptations associated with the unusual reproductive strategy in these mollusks. There has been great interest in studying reproduction-related proteins as such studies may not only answer fundamental questions about speciation and evolution, but also solve practical problems of animal infertility and pest outbreak. Our study has demonstrated the effectiveness of an integrated proteomic and transcriptomic approach in understanding the heavy maternal investment of proteins in the eggs of a non-model snail, and how the reproductive proteins may have evolved during the transition from laying underwater eggs to aerial eggs. Copyright © 2017 Elsevier B.V. All rights reserved.

Oxidative-stress-related proteome changes in Helicobacter pylori-infected human gastric mucosa.

PubMed Central

Baek, Hye Yeon; Lim, Joo Weon; Kim, Hyeyoung; Kim, Jung Mogg; Kim, Joo Sung; Jung, Hyun Chae; Kim, Kyung Hwan

2004-01-01

Helicobacter pylori infection leads to gastroduodenal inflammation, peptic ulceration and gastric carcinoma. Proteomic analysis of the human gastric mucosa from the patients with erosive gastritis, peptic ulcer or gastric cancer, which were either infected or not with H. pylori, was used to determine the differentially expressed proteins by H. pylori in the human gastric mucosa in order to investigate the pathogenic mechanism of H. pylori -induced gastric diseases. Prior to the experiment, the expression of the main 18 proteins were identified in the gastric mucosa and used for a proteome map of the human gastric mucosa. Using two-dimensional electrophoresis of the protein isolated from the H. pylori -infected tissues, Coomassie Brilliant Blue staining and computerized analysis of the stained gel, the expression of eight proteins were altered in the H. pylori -infected tissues compared with the non-infected tissues. MS analysis (matrix-assisted laser desorption/ionization-time of flight MS) of the tryptic fragment and a data search allowed the the identification of the four increased proteins (78 kDa glucose-regulated protein precursor, endoplasmin precursor, aldehyde dehydrogenase 2 and L-lactate dehydrogenase B chain) and the four decreased proteins (intracellular chloride channel protein 1, glutathione S-transferase, heat-shock protein 60 and cytokeratin 8) caused by H. pylori infection in the gastric mucosa. These proteins are related to cell proliferation, carcinogenesis, cytoskeletal function and cellular defence mechanism. The common feature is that these proteins are related to oxidative-stress-mediated cell damage. In conclusion, the established gastric mucosal proteome map might be useful for detecting the disease-related protein changes. The H. pylori -induced alterations in protein expression demonstrate the involvement of oxidative stress in the pathogenesis of H. pylori -induced gastric diseases, including inflammation, ulceration and carcinogenesis. PMID:14711373

Serum biomarkers of Burkholderia mallei infection elucidated by proteomic imaging of skin and lung abscesses.

PubMed

Glaros, Trevor G; Blancett, Candace D; Bell, Todd M; Natesan, Mohan; Ulrich, Robert G

2015-01-01

The bacterium Burkholderia mallei is the etiological agent of glanders, a highly contagious, often fatal zoonotic infectious disease that is also a biodefense concern. Clinical laboratory assays that analyze blood or other biological fluids are the highest priority because these specimens can be collected with minimal risk to the patient. However, progress in developing sensitive assays for monitoring B. mallei infection is hampered by a shortage of useful biomarkers. Reasoning that there should be a strong correlation between the proteomes of infected tissues and circulating serum, we employed imaging mass spectrometry (IMS) of thin-sectioned tissues from Chlorocebus aethiops (African green) monkeys infected with B. mallei to localize host and pathogen proteins that were associated with abscesses. Using laser-capture microdissection of specific regions identified by IMS and histology within the tissue sections, a more extensive proteomic analysis was performed by a technique that combined the physical separation capabilities of liquid chromatography (LC) with the sensitive mass analysis capabilities of mass spectrometry (LC-MS/MS). By examining standard formalin-fixed, paraffin-embedded tissue sections, this strategy resulted in the identification of several proteins that were associated with lung and skin abscesses, including the host protein calprotectin and the pathogen protein GroEL. Elevated levels of calprotectin detected by ELISA and antibody responses to GroEL, measured by a microarray of the bacterial proteome, were subsequently detected in the sera of C. aethiops, Macaca mulatta, and Macaca fascicularis primates infected with B. mallei. Our results demonstrate that a combination of multidimensional MS analysis of traditional histology specimens with high-content protein microarrays can be used to discover lead pairs of host-pathogen biomarkers of infection that are identifiable in biological fluids.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng, E-mail: yinsheng.wang@ucr.edu

Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 humanmore » skin fibroblasts following acute MMA(III) exposure. Among the ∼ 6500 unique proteins quantified, ∼ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. - Highlights: • MMA(III)-induced perturbation of the entire proteome of GM00637 cells is studied. • Quantitative proteomic approach revealed alterations of multiple cellular pathways. • MMA(III) inhibits de novo cholesterol biosynthesis. • MMA(III) perturbs Nrf2 pathway and selenoprotein synthesis.« less

Redox proteomic analysis of serum from aortic anerurysm patients: insights on oxidation of specific protein target.

PubMed

Spadaccio, Cristiano; Coccia, Raffaella; Perluigi, Marzia; Pupo, Gilda; Schininà, Maria Eugenia; Giorgi, Alessandra; Blarzino, Carla; Nappi, Francesco; Sutherland, Fraser W; Chello, Massimo; Di Domenico, Fabio

2016-06-21

oxidative stress is undoubtedly one of the main players in abdominal aortic aneurysm (AAA) pathophysiology. Recent studies in AAA patients reported an increase in the indices of oxidative damage at the tissue level and in biological fluids coupled with the loss of counter-regulatory mechanisms of protection from oxidative stress. We recently reported, in a proteomic analysis of AAA patient sera, changes in the expression of several proteins exerting important modulatory activities on cellular proliferation, differentiation and response to damage. This study aimed to explore the involvement of protein oxidation, at peripheral levels, in AAA. a redox proteomic approach was used to investigate total and specific protein carbonylation and protein-bound 4-hydroxy-2-nonenal (HNE) in the serum of AAA patients compared with age-matched controls. our results show increased oxidative damage to protein as indexed by the total carbonyl levels and total protein-bound HNE. By redox proteomics we identified specific carbonylation of three serum proteins: serum retinol-binding protein, vitamin D-binding protein and fibrinogen α-chain HNE. We also identified increased protein-bound HNE levels for hemopexin, IgK chain C region and IgK chain V-III region SIE. In addition we found a high correlation between specific protein carbonylation and protein-bound HNE and the aortic diameter. Moreover the analysis of serum proteins with antioxidant activity demonstrates the oxidation of albumin together with the overexpression of transferrin, haptoglobin and HSPs 90, 70, 60 and 32. this study support the involvement of oxidative stress in the pathogenesis of AAA and might provide a further degree of knowledge in the cause-effect role of oxidative stress shedding new light on the molecular candidates involved in the disease.

NCI's Proteome Characterization Centers Announced | Office of Cancer Clinical Proteomics Research

Cancer.gov

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.

Proteomics in medical microbiology.

PubMed

Cash, P

2000-04-01

The techniques of proteomics (high resolution two-dimensional electrophoresis and protein characterisation) are widely used for microbiological research to analyse global protein synthesis as an indicator of gene expression. The rapid progress in microbial proteomics has been achieved through the wide availability of whole genome sequences for a number of bacterial groups. Beyond providing a basic understanding of microbial gene expression, proteomics has also played a role in medical areas of microbiology. Progress has been made in the use of the techniques for investigating the epidemiology and taxonomy of human microbial pathogens, the identification of novel pathogenic mechanisms and the analysis of drug resistance. In each of these areas, proteomics has provided new insights that complement genomic-based investigations. This review describes the current progress in these research fields and highlights some of the technical challenges existing for the application of proteomics in medical microbiology. The latter concern the analysis of genetically heterogeneous bacterial populations and the integration of the proteomic and genomic data for these bacteria. The characterisation of the proteomes of bacterial pathogens growing in their natural hosts remains a future challenge.

Proteomic analysis of ligamentum flavum from patients with lumbar spinal stenosis.

PubMed

Kamita, Masahiro; Mori, Taiki; Sakai, Yoshihito; Ito, Sadayuki; Gomi, Masahiro; Miyamoto, Yuko; Harada, Atsushi; Niida, Shumpei; Yamada, Tesshi; Watanabe, Ken; Ono, Masaya

2015-05-01

Lumbar spinal stenosis (LSS) is a syndromic degenerative spinal disease and is characterized by spinal canal narrowing with subsequent neural compression causing gait disturbances. Although LSS is a major age-related musculoskeletal disease that causes large decreases in the daily living activities of the elderly, its molecular pathology has not been investigated using proteomics. Thus, we used several proteomic technologies to analyze the ligamentum flavum (LF) of individuals with LSS. Using comprehensive proteomics with strong cation exchange fractionation, we detected 1288 proteins in these LF samples. A GO analysis of the comprehensive proteome revealed that more than 30% of the identified proteins were extracellular. Next, we used 2D image converted analysis of LC/MS to compare LF obtained from individuals with LSS to that obtained from individuals with disc herniation (nondegenerative control). We detected 64 781 MS peaks and identified 1675 differentially expressed peptides derived from 286 proteins. We verified four differentially expressed proteins (fibronectin, serine protease HTRA1, tenascin, and asporin) by quantitative proteomics using SRM/MRM. The present proteomic study is the first to identify proteins from degenerated and hypertrophied LF in LSS, which will help in studying LSS. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Label-free proteomic analysis to confirm the predicted proteome of Corynebacterium pseudotuberculosis under nitrosative stress mediated by nitric oxide.

PubMed

Silva, Wanderson M; Carvalho, Rodrigo D; Soares, Siomar C; Bastos, Isabela Fs; Folador, Edson L; Souza, Gustavo Hmf; Le Loir, Yves; Miyoshi, Anderson; Silva, Artur; Azevedo, Vasco

2014-12-04

Corynebacterium pseudotuberculosis biovar ovis is a facultative intracellular pathogen, and the etiological agent of caseous lymphadenitis in small ruminants. During the infection process, the bacterium is subjected to several stress conditions, including nitrosative stress, which is caused by nitric oxide (NO). In silico analysis of the genome of C. pseudotuberculosis ovis 1002 predicted several genes that could influence the resistance of this pathogen to nitrosative stress. Here, we applied high-throughput proteomics using high definition mass spectrometry to characterize the functional genome of C. pseudotuberculosis ovis 1002 in the presence of NO-donor Diethylenetriamine/nitric oxide adduct (DETA/NO), with the aim of identifying proteins involved in nitrosative stress resistance. We characterized 835 proteins, representing approximately 41% of the predicted proteome of C. pseudotuberculosis ovis 1002, following exposure to nitrosative stress. In total, 102 proteins were exclusive to the proteome of DETA/NO-induced cells, and a further 58 proteins were differentially regulated between the DETA/NO and control conditions. An interactomic analysis of the differential proteome of C. pseudotuberculosis in response to nitrosative stress was also performed. Our proteomic data set suggested the activation of both a general stress response and a specific nitrosative stress response, as well as changes in proteins involved in cellular metabolism, detoxification, transcriptional regulation, and DNA synthesis and repair. Our proteomic analysis validated previously-determined in silico data for C. pseudotuberculosis ovis 1002. In addition, proteomic screening performed in the presence of NO enabled the identification of a set of factors that can influence the resistance and survival of C. pseudotuberculosis during exposure to nitrosative stress.

CPTAC Prospective Biospecimen Collection Solicitation | Office of Cancer Clinical Proteomics Research

Cancer.gov

A new funding opportunity in support of the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) seeks to prospectively procure tumor samples, collected for proteomics investigation.

Proteomics: a new approach to the study of disease.

PubMed

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.

Birth of plant proteomics in India: a new horizon.

PubMed

Narula, Kanika; Pandey, Aarti; Gayali, Saurabh; Chakraborty, Niranjan; Chakraborty, Subhra

2015-09-08

In the post-genomic era, proteomics is acknowledged as the next frontier for biological research. Although India has a long and distinguished tradition in protein research, the initiation of proteomics studies was a new horizon. Protein research witnessed enormous progress in protein separation, high-resolution refinements, biochemical identification of the proteins, protein-protein interaction, and structure-function analysis. Plant proteomics research, in India, began its journey on investigation of the proteome profiling, complexity analysis, protein trafficking, and biochemical modeling. The research article by Bhushan et al. in 2006 marked the birth of the plant proteomics research in India. Since then plant proteomics studies expanded progressively and are now being carried out in various institutions spread across the country. The compilation presented here seeks to trace the history of development in the area during the past decade based on publications till date. In this review, we emphasize on outcomes of the field providing prospects on proteomic pathway analyses. Finally, we discuss the connotation of strategies and the potential that would provide the framework of plant proteome research. The past decades have seen rapidly growing number of sequenced plant genomes and associated genomic resources. To keep pace with this increasing body of data, India is in the provisional phase of proteomics research to develop a comparative hub for plant proteomes and protein families, but it requires a strong impetus from intellectuals, entrepreneurs, and government agencies. Here, we aim to provide an overview of past, present and future of Indian plant proteomics, which would serve as an evaluation platform for those seeking to incorporate proteomics into their research programs. This article is part of a Special Issue entitled: Proteomics in India. Copyright © 2015 Elsevier B.V. All rights reserved.

Pressurized Pepsin Digestion in Proteomics

PubMed Central

López-Ferrer, Daniel; Petritis, Konstantinos; Robinson, Errol W.; Hixson, Kim K.; Tian, Zhixin; Lee, Jung Hwa; Lee, Sang-Won; Tolić, Nikola; Weitz, Karl K.; Belov, Mikhail E.; Smith, Richard D.; Paša-Tolić, Ljiljana

2011-01-01

Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome. PMID:20627868

Detailed Functional and Proteomic Characterization of Fludarabine Resistance in Mantle Cell Lymphoma Cells

PubMed Central

Lorkova, Lucie; Scigelova, Michaela; Arrey, Tabiwang Ndipanquang; Vit, Ondrej; Pospisilova, Jana; Doktorova, Eliska; Klanova, Magdalena; Alam, Mahmudul; Vockova, Petra; Maswabi, Bokang

2015-01-01

Mantle cell lymphoma (MCL) is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR) and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino). We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine) and to an inhibitor of Bruton tyrosine kinase (BTK) ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib) or remained unaffacted (cisplatin, bendamustine). The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK) and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib), but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies. PMID:26285204

NCI Launches Proteomics Assay Portal | Office of Cancer Clinical Proteomics Research

Cancer.gov

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

CPTAC Releases Largest-Ever Colorectal Cancer Proteome Dataset from Previously Genome Characterized Tumors | Office of Cancer Clinical Proteomics Research

Cancer.gov

On September 4, 2013, NCI’s Clinical Proteomics Tumor Analysis Consortium (CPTAC) publicly released proteomic data produced from colorectal tumor samples previously analyzed by The Cancer Genome Atlas (TCGA).  This is the initial release of proteomic tumor data designed to complement genomic data on the same tumors. The data is publicly available at the CPTAC data portal.

Deep sequencing and proteomic analysis of the microRNA-induced silencing complex in human red blood cells.

PubMed

Azzouzi, Imane; Moest, Hansjoerg; Wollscheid, Bernd; Schmugge, Markus; Eekels, Julia J M; Speer, Oliver

2015-05-01

During maturation, erythropoietic cells extrude their nuclei but retain their ability to respond to oxidant stress by tightly regulating protein translation. Several studies have reported microRNA-mediated regulation of translation during terminal stages of erythropoiesis, even after enucleation. In the present study, we performed a detailed examination of the endogenous microRNA machinery in human red blood cells using a combination of deep sequencing analysis of microRNAs and proteomic analysis of the microRNA-induced silencing complex. Among the 197 different microRNAs detected, miR-451a was the most abundant, representing more than 60% of all read sequences. In addition, miR-451a and its known target, 14-3-3ζ mRNA, were bound to the microRNA-induced silencing complex, implying their direct interaction in red blood cells. The proteomic characterization of endogenous Argonaute 2-associated microRNA-induced silencing complex revealed 26 cofactor candidates. Among these cofactors, we identified several RNA-binding proteins, as well as motor proteins and vesicular trafficking proteins. Our results demonstrate that red blood cells contain complex microRNA machinery, which might enable immature red blood cells to control protein translation independent of de novo nuclei information. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Protein content and functional characteristics of serum-purified exosomes from patients with colorectal cancer revealed by quantitative proteomics.

PubMed

Chen, Yanyu; Xie, Yong; Xu, Lai; Zhan, Shaohua; Xiao, Yi; Gao, Yanpan; Wu, Bin; Ge, Wei

2017-02-15

Tumor cells of colorectal cancer (CRC) release exosomes into the circulation. These exosomes can mediate communication between cells and affect various tumor-related processes in their target cells. We present a quantitative proteomics analysis of the exosomes purified from serum of patients with CRC and normal volunteers; data are available via ProteomeXchange with identifier PXD003875. We identified 918 proteins with an overlap of 725 Gene IDs in the Exocarta proteins list. Compared with the serum-purified exosomes (SPEs) of normal volunteers, we found 36 proteins upregulated and 22 proteins downregulated in the SPEs of CRC patients. Bioinformatics analysis revealed that upregulated proteins are involved in processes that modulate the pretumorigenic microenvironment for metastasis. In contrast, differentially expressed proteins (DEPs) that play critical roles in tumor growth and cell survival were principally downregulated. Our study demonstrates that SPEs of CRC patients play a pivotal role in promoting the tumor invasiveness, but have minimal influence on putative alterations in tumor survival or proliferation. According to bioinformatics analysis, we speculate that the protein contents of exosomes might be associated with whether they are involved in premetastatic niche establishment or growth and survival of metastatic tumor cells. This information will be helpful in elucidating the pathophysiological functions of tumor-derived exosomes, and aid in the development of CRC diagnostics and therapeutics. © 2016 UICC.

A Simple Method for Rapid Depletion of Rubisco from Soybean (Glycine max) Leaf for Proteomic Analysis of Lower Abundance Proteins

USDA-ARS?s Scientific Manuscript database

2-DE analysis of complex plant proteomes has limited dynamic resolution because only abundant proteins can be detected. Proteomic assessment of the low abundance proteins within leaf tissue is difficult when it is comprised of 30 – 50% of the CO2 fixation enzyme Rubisco. Resolution can be improved t...

Influence of Diet on the Proteome of Drosophila Melanogaster as Assessed by Two-Dimensional Gel Electrophoresis and Capillary Liquid Chromatography–Mass Spectrometry: The Hamburger Effect Revisited

PubMed Central

Culwell, Thomas F.; Thulin, Craig D.; Merrell, Karen J.; Graves, Steven W.

2008-01-01

Proteomic biomarker discovery has been called into question. Diamandis hypothesized that seemingly trivial factors, such as eating a hamburger, may cause sufficient proteomic change as to confound proteomic differences. This has been termed the hamburger effect. Little is known about the variability of complex proteomes in response to the environment. Two methods—two-dimensional gel electrophoresis (2DGE) and capillary liquid chromatography–electrospray ionization time-of-flight mass spectrometry (LCMS)—were used to study the hamburger effect in two cross-sections of the soluble fruit fly proteome. 2DGE measured abundant proteins, whereas LCMS measured small proteins and peptides. Proteomic differences between males and females were first evaluated to assess the discriminatory capability of the methods. Likewise, wild-type and white-eyed flies were analyzed as a further demonstration that genetically based proteomic differences could be observed above the background analytical variation. Then dietary interventions were imposed. Ethanol was added to the diet of some populations without significant proteomic effect. However, after a 24-h fast, proteomic differences were found using LCMS but not 2DGE. Even so, only three of ~1000 molecular species were altered significantly, suggesting that the influence of even an extreme diet change produced only modest proteomic variability, and that much of the fruit fly proteome remains relatively constant in response to diet. These experiments suggest that proteomics can be a viable approach to biomarker discovery. PMID:19137114

Pressurized Pepsin Digestion in Proteomics: An Automatable Alternative to Trypsin for Integrated Top-down Bottom-up Proteomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lopez-Ferrer, Daniel; Petritis, Konstantinos; Robinson, Errol W.

2011-02-01

Integrated top-down bottom-up proteomics combined with online digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to highthroughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications (PTMs). Herein, we describe recent efforts towards efficient integration of bottom-up and top-down LCMS based proteomic strategies. Since most proteomic platforms (i.e. LC systems) operate in acidic environments, we exploited themore » compatibility of the pepsin (i.e. the enzyme’s natural acidic activity) for the integration of bottom-up and top-down proteomics. Pressure enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an offline mode using a Barocycler or an online mode using a modified high pressure LC system referred to as a fast online digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultra-rapid integrated bottom-up top-down proteomic strategy employing a standard mixture of proteins and a monkey pox virus proteome.« less

The Role of Translational Regulation in Survival after Radiation Damage; an Opportunity for Proteomics Analysis

PubMed Central

Stickel, Stefanie; Gomes, Nathan; Su, Tin Tin

2014-01-01

In this review, we will summarize the data from different model systems that illustrate the need for proteome-wide analyses of the biological consequences of ionizing radiation (IR). IR remains one of three main therapy choices for oncology, the others being surgery and chemotherapy. Understanding how cells and tissues respond to IR is essential for improving therapeutic regimes against cancer. Numerous studies demonstrating the changes in the transcriptome following exposure to IR, in diverse systems, can be found in the scientific literature. However, the limitation of our knowledge is illustrated by the fact that the number of transcripts that change after IR exposure is approximately an order of magnitude lower than the number of transcripts that re-localize to or from ribosomes under similar conditions. Furthermore, changes in the post-translational modifications of proteins (phosphorylation, acetylation as well as degradation) are profoundly important for the cellular response to IR. These considerations make proteomics a highly suitable tool for mechanistic studies of the effect of IR. Strikingly such studies remain outnumbered by those utilizing proteomics for diagnostic purposes such as the identification of biomarkers for the outcome of radiation therapy. Here we will discuss the role of the ribosome and translational regulation in the survival and preservation of cells and tissues after exposure to ionizing radiation. In doing so we hope to provide a strong incentive for the study of proteome-wide changes following IR exposure. PMID:26269784

Xylem sap proteomics.

PubMed

de Bernonville, Thomas Dugé; Albenne, Cécile; Arlat, Matthieu; Hoffmann, Laurent; Lauber, Emmanuelle; Jamet, Elisabeth

2014-01-01

Proteomic analysis of xylem sap has recently become a major field of interest to understand several biological questions related to plant development and responses to environmental clues. The xylem sap appears as a dynamic fluid undergoing changes in its proteome upon abiotic and biotic stresses. Unlike cell compartments which are amenable to purification in sufficient amount prior to proteomic analysis, the xylem sap has to be collected in particular conditions to avoid contamination by intracellular proteins and to obtain enough material. A model plant like Arabidopsis thaliana is not suitable for such an analysis because efficient harvesting of xylem sap is difficult. The analysis of the xylem sap proteome also requires specific procedures to concentrate proteins and to focus on proteins predicted to be secreted. Indeed, xylem sap proteins appear to be synthesized and secreted in the root stele or to originate from dying differentiated xylem cells. This chapter describes protocols to collect xylem sap from Brassica species and to prepare total and N-glycoprotein extracts for identification of proteins by mass spectrometry analyses and bioinformatics.

Extreme diversity of scorpion venom peptides and proteins revealed by transcriptomic analysis: implication for proteome evolution of scorpion venom arsenal.

PubMed

Ma, Yibao; He, Yawen; Zhao, Ruiming; Wu, Yingliang; Li, Wenxin; Cao, Zhijian

2012-02-16

Venom is an important genetic development crucial to the survival of scorpions for over 400 million years. We studied the evolution of the scorpion venom arsenal by means of comparative transcriptome analysis of venom glands and phylogenetic analysis of shared types of venom peptides and proteins between buthids and euscorpiids. Fifteen types of venom peptides and proteins were sequenced during the venom gland transcriptome analyses of two Buthidae species (Lychas mucronatus and Isometrus maculatus) and one Euscorpiidae species (Scorpiops margerisonae). Great diversity has been observed in translated amino acid sequences of these transcripts for venom peptides and proteins. Seven types of venom peptides and proteins were shared between buthids and euscorpiids. Molecular phylogenetic analysis revealed that at least five of the seven common types of venom peptides and proteins were likely recruited into the scorpion venom proteome before the lineage split between Buthidae and Euscorpiidae with their corresponding genes undergoing individual or multiple gene duplication events. These are α-KTxs, βKSPNs (β-KTxs and scorpines), anionic peptides, La1-like peptides, and SPSVs (serine proteases from scorpion venom). Multiple types of venom peptides and proteins were demonstrated to be continuously recruited into the venom proteome during the evolution process of individual scorpion lineages. Our results provide an insight into the recruitment pattern of the scorpion venom arsenal for the first time. Copyright © 2011 Elsevier B.V. All rights reserved.

Three dimensional liquid chromatography coupling ion exchange chromatography/hydrophobic interaction chromatography/reverse phase chromatography for effective protein separation in top-down proteomics.

PubMed

Valeja, Santosh G; Xiu, Lichen; Gregorich, Zachery R; Guner, Huseyin; Jin, Song; Ge, Ying

2015-01-01

To address the complexity of the proteome in mass spectrometry (MS)-based top-down proteomics, multidimensional liquid chromatography (MDLC) strategies that can effectively separate proteins with high resolution and automation are highly desirable. Although various MDLC methods that can effectively separate peptides from protein digests exist, very few MDLC strategies, primarily consisting of 2DLC, are available for intact protein separation, which is insufficient to address the complexity of the proteome. We recently demonstrated that hydrophobic interaction chromatography (HIC) utilizing a MS-compatible salt can provide high resolution separation of intact proteins for top-down proteomics. Herein, we have developed a novel 3DLC strategy by coupling HIC with ion exchange chromatography (IEC) and reverse phase chromatography (RPC) for intact protein separation. We demonstrated that a 3D (IEC-HIC-RPC) approach greatly outperformed the conventional 2D IEC-RPC approach. For the same IEC fraction (out of 35 fractions) from a crude HEK 293 cell lysate, a total of 640 proteins were identified in the 3D approach (corresponding to 201 nonredundant proteins) as compared to 47 in the 2D approach, whereas simply prolonging the gradients in RPC in the 2D approach only led to minimal improvement in protein separation and identifications. Therefore, this novel 3DLC method has great potential for effective separation of intact proteins to achieve deep proteome coverage in top-down proteomics.

Dynamic regulation of hepatic lipid droplet properties by diet.

PubMed

Crunk, Amanda E; Monks, Jenifer; Murakami, Aya; Jackman, Matthew; Maclean, Paul S; Ladinsky, Mark; Bales, Elise S; Cain, Shannon; Orlicky, David J; McManaman, James L

2013-01-01

Cytoplasmic lipid droplets (CLD) are organelle-like structures that function in neutral lipid storage, transport and metabolism through the actions of specific surface-associated proteins. Although diet and metabolism influence hepatic CLD levels, how they affect CLD protein composition is largely unknown. We used non-biased, shotgun, proteomics in combination with metabolic analysis, quantitative immunoblotting, electron microscopy and confocal imaging to define the effects of low- and high-fat diets on CLD properties in fasted-refed mice. We found that the hepatic CLD proteome is distinct from that of CLD from other mammalian tissues, containing enzymes from multiple metabolic pathways. The hepatic CLD proteome is also differentially affected by dietary fat content and hepatic metabolic status. High fat feeding markedly increased the CLD surface density of perilipin-2, a critical regulator of hepatic neutral lipid storage, whereas it reduced CLD levels of betaine-homocysteine S-methyltransferase, an enzyme regulator of homocysteine levels linked to fatty liver disease and hepatocellular carcinoma. Collectively our data demonstrate that the hepatic CLD proteome is enriched in metabolic enzymes, and that it is qualitatively and quantitatively regulated by diet and metabolism. These findings implicate CLD in the regulation of hepatic metabolic processes, and suggest that their properties undergo reorganization in response to hepatic metabolic demands.

Proteomic changes during intestinal cell maturation in vivo

PubMed Central

Chang, Jinsook; Chance, Mark R.; Nicholas, Courtney; Ahmed, Naseem; Guilmeau, Sandra; Flandez, Marta; Wang, Donghai; Byun, Do-Sun; Nasser, Shannon; Albanese, Joseph M.; Corner, Georgia A.; Heerdt, Barbara G.; Wilson, Andrew J.; Augenlicht, Leonard H.; Mariadason, John M.

2008-01-01

Intestinal epithelial cells undergo progressive cell maturation as they migrate along the crypt-villus axis. To determine molecular signatures that define this process, proteins differentially expressed between the crypt and villus were identified by 2D-DIGE and MALDI-MS. Forty-six differentially expressed proteins were identified, several of which were validated by immunohistochemistry. Proteins upregulated in the villus were enriched for those involved in brush border assembly and lipid uptake, established features of differentiated intestinal epithelial cells. Multiple proteins involved in glycolysis were also upregulated in the villus, suggesting increased glycolysis is a feature of intestinal cell differentiation. Conversely, proteins involved in nucleotide metabolism, and protein processing and folding were increased in the crypt, consistent with functions associated with cell proliferation. Three novel paneth cell markers, AGR2, HSPA5 and RRBP1 were also identified. Notably, significant correlation was observed between overall proteomic changes and corresponding gene expression changes along the crypt-villus axis, indicating intestinal cell maturation is primarily regulated at the transcriptional level. This proteomic profiling analysis identified several novel proteins and functional processes differentially induced during intestinal cell maturation in vivo. Integration of proteomic, immunohistochemical, and parallel gene expression datasets demonstrate the coordinated manner in which intestinal cell maturation is regulated. PMID:18824147

Dynamic Regulation of Hepatic Lipid Droplet Properties by Diet

PubMed Central

Crunk, Amanda E.; Monks, Jenifer; Murakami, Aya; Jackman, Matthew; MacLean, Paul S.; Ladinsky, Mark; Bales, Elise S.; Cain, Shannon; Orlicky, David J.; McManaman, James L.

2013-01-01

Cytoplasmic lipid droplets (CLD) are organelle-like structures that function in neutral lipid storage, transport and metabolism through the actions of specific surface-associated proteins. Although diet and metabolism influence hepatic CLD levels, how they affect CLD protein composition is largely unknown. We used non-biased, shotgun, proteomics in combination with metabolic analysis, quantitative immunoblotting, electron microscopy and confocal imaging to define the effects of low- and high-fat diets on CLD properties in fasted-refed mice. We found that the hepatic CLD proteome is distinct from that of CLD from other mammalian tissues, containing enzymes from multiple metabolic pathways. The hepatic CLD proteome is also differentially affected by dietary fat content and hepatic metabolic status. High fat feeding markedly increased the CLD surface density of perilipin-2, a critical regulator of hepatic neutral lipid storage, whereas it reduced CLD levels of betaine-homocysteine S-methyltransferase, an enzyme regulator of homocysteine levels linked to fatty liver disease and hepatocellular carcinoma. Collectively our data demonstrate that the hepatic CLD proteome is enriched in metabolic enzymes, and that it is qualitatively and quantitatively regulated by diet and metabolism. These findings implicate CLD in the regulation of hepatic metabolic processes, and suggest that their properties undergo reorganization in response to hepatic metabolic demands. PMID:23874434

Cloud CPFP: a shotgun proteomics data analysis pipeline using cloud and high performance computing.

PubMed

Trudgian, David C; Mirzaei, Hamid

2012-12-07

We have extended the functionality of the Central Proteomics Facilities Pipeline (CPFP) to allow use of remote cloud and high performance computing (HPC) resources for shotgun proteomics data processing. CPFP has been modified to include modular local and remote scheduling for data processing jobs. The pipeline can now be run on a single PC or server, a local cluster, a remote HPC cluster, and/or the Amazon Web Services (AWS) cloud. We provide public images that allow easy deployment of CPFP in its entirety in the AWS cloud. This significantly reduces the effort necessary to use the software, and allows proteomics laboratories to pay for compute time ad hoc, rather than obtaining and maintaining expensive local server clusters. Alternatively the Amazon cloud can be used to increase the throughput of a local installation of CPFP as necessary. We demonstrate that cloud CPFP allows users to process data at higher speed than local installations but with similar cost and lower staff requirements. In addition to the computational improvements, the web interface to CPFP is simplified, and other functionalities are enhanced. The software is under active development at two leading institutions and continues to be released under an open-source license at http://cpfp.sourceforge.net.

Blood coagulation abnormalities in multibacillary leprosy patients.

PubMed

Silva, Débora Santos da; Teixeira, Lisandra Antonia Castro; Beghini, Daniela Gois; Ferreira, André Teixeira da Silva; Pinho, Márcia de Berredo Moreira; Rosa, Patricia Sammarco; Ribeiro, Marli Rambaldi; Freire, Monica Di Calafiori; Hacker, Mariana Andrea; Nery, José Augusto da Costa; Pessolani, Maria Cristina Vidal; Tovar, Ana Maria Freire; Sarno, Euzenir Nunes; Perales, Jonas; Bozza, Fernando Augusto; Esquenazi, Danuza; Monteiro, Robson Queiroz; Lara, Flavio Alves

2018-03-01

Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae infection. In 2016, more than 200,000 new cases of leprosy were detected around the world, representing the most frequent cause of infectious irreversible deformities and disabilities. In the present work, we demonstrate a consistent procoagulant profile on 40 reactional and non-reactional multibacillary leprosy patients. A retrospective analysis in search of signs of coagulation abnormalities among 638 leprosy patients identified 35 leprosy patients (5.48%) which displayed a characteristic lipid-like clot formed between blood clot and serum during serum harvesting, herein named 'leprosum clot'. Most of these patients (n = 16, 45.7%) belonged to the lepromatous leprosy pole of the disease. In addition, formation of the leprosum clot was directly correlated with increased plasma levels of soluble tissue factor and von Willebrand factor. High performance thin layer chromatography demonstrated a high content of neutral lipids in the leprosum clot, and proteomic analysis demonstrated that the leprosum clot presented in these patients is highly enriched in fibrin. Remarkably, differential 2D-proteomics analysis between leprosum clots and control clots identified two proteins present only in leprosy patients clots: complement component 3 and 4 and inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP). In agreement with those observations we demonstrated that M. leprae induces hepatocytes release of IHRP in vitro. We demonstrated that leprosy MB patients develop a procoagulant status due to high levels of plasmatic fibrinogen, anti-cardiolipin antibodies, von Willebrand factor and soluble tissue factor. We propose that some of these components, fibrinogen for example, presents potential as predictive biomarkers of leprosy reactions, generating tools for earlier diagnosis and treatment of these events.

Proteomics of effector-triggered immunity (ETI) in plants.

PubMed

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.

Proteomics analyses for the global proteins in the brain tissues of different human prion diseases.

PubMed

Shi, Qi; Chen, Li-Na; Zhang, Bao-Yun; Xiao, Kang; Zhou, Wei; Chen, Cao; Zhang, Xiao-Mei; Tian, Chan; Gao, Chen; Wang, Jing; Han, Jun; Dong, Xiao-Ping

2015-04-01

Proteomics changes of brain tissues have been described in different neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. However, the brain proteomics of human prion disease remains less understood. In the study, the proteomics patterns of cortex and cerebellum of brain tissues of sporadic Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD were analyzed with isobaric tags for relative and absolute quantitation combined with multidimensional liquid chromatography and MS analysis, with the brains from three normal individuals as controls. Global protein profiling, significant pathway, and functional categories were analyzed. In total, 2287 proteins were identified with quantitative information both in cortex and cerebellum regions. Cerebellum tissues appeared to contain more up- and down-regulated proteins (727 proteins) than cortex regions (312 proteins) of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD. Viral myocarditis, Parkinson's disease, Alzheimer's disease, lysosome, oxidative phosphorylation, protein export, and drug metabolism-cytochrome P450 were the most commonly affected pathways of the three kinds of diseases. Almost coincident biological functions were identified in the brain tissues of the three diseases. In all, data here demonstrate that the brain tissues of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD have obvious proteomics changes at their terminal stages, which show the similarities not only among human prion diseases but also with other neurodegeneration diseases. This is the first study to provide a reference proteome map for human prion diseases and will be helpful for future studies focused on potential biomarkers for the diagnosis and therapy of human prion diseases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Redox Proteomics in Selected Neurodegenerative Disorders: From Its Infancy to Future Applications

PubMed Central

Perluigi, Marzia; Reed, Tanea; Muharib, Tasneem; Hughes, Christopher P.; Robinson, Renã A.S.; Sultana, Rukhsana

2012-01-01

Abstract Several studies demonstrated that oxidative damage is a characteristic feature of many neurodegenerative diseases. The accumulation of oxidatively modified proteins may disrupt cellular functions by affecting protein expression, protein turnover, cell signaling, and induction of apoptosis and necrosis, suggesting that protein oxidation could have both physiological and pathological significance. For nearly two decades, our laboratory focused particular attention on studying oxidative damage of proteins and how their chemical modifications induced by reactive oxygen species/reactive nitrogen species correlate with pathology, biochemical alterations, and clinical presentations of Alzheimer's disease. This comprehensive article outlines basic knowledge of oxidative modification of proteins and lipids, followed by the principles of redox proteomics analysis, which also involve recent advances of mass spectrometry technology, and its application to selected age-related neurodegenerative diseases. Redox proteomics results obtained in different diseases and animal models thereof may provide new insights into the main mechanisms involved in the pathogenesis and progression of oxidative-stress-related neurodegenerative disorders. Redox proteomics can be considered a multifaceted approach that has the potential to provide insights into the molecular mechanisms of a disease, to find disease markers, as well as to identify potential targets for drug therapy. Considering the importance of a better understanding of the cause/effect of protein dysfunction in the pathogenesis and progression of neurodegenerative disorders, this article provides an overview of the intrinsic power of the redox proteomics approach together with the most significant results obtained by our laboratory and others during almost 10 years of research on neurodegenerative disorders since we initiated the field of redox proteomics. Antioxid. Redox Signal. 17, 1610–1655. PMID:22115501

Design and analysis issues in quantitative proteomics studies.

PubMed

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.

Establishing Substantial Equivalence: Proteomics

NASA Astrophysics Data System (ADS)

Lovegrove, Alison; Salt, Louise; Shewry, Peter R.

Wheat is a major crop in world agriculture and is consumed after processing into a range of food products. It is therefore of great importance to determine the consequences (intended and unintended) of transgenesis in wheat and whether genetically modified lines are substantially equivalent to those produced by conventional plant breeding. Proteomic analysis is one of several approaches which can be used to address these questions. Two-dimensional PAGE (2D PAGE) remains the most widely available method for proteomic analysis, but is notoriously difficult to reproduce between laboratories. We therefore describe methods which have been developed as standard operating procedures in our laboratory to ensure the reproducibility of proteomic analyses of wheat using 2D PAGE analysis of grain proteins.

Integrated Analysis of Transcriptomic and Proteomic Data

PubMed Central

Haider, Saad; Pal, Ranadip

2013-01-01

Until recently, understanding the regulatory behavior of cells has been pursued through independent analysis of the transcriptome or the proteome. Based on the central dogma, it was generally assumed that there exist a direct correspondence between mRNA transcripts and generated protein expressions. However, recent studies have shown that the correlation between mRNA and Protein expressions can be low due to various factors such as different half lives and post transcription machinery. Thus, a joint analysis of the transcriptomic and proteomic data can provide useful insights that may not be deciphered from individual analysis of mRNA or protein expressions. This article reviews the existing major approaches for joint analysis of transcriptomic and proteomic data. We categorize the different approaches into eight main categories based on the initial algorithm and final analysis goal. We further present analogies with other domains and discuss the existing research problems in this area. PMID:24082820

freeQuant: A Mass Spectrometry Label-Free Quantification Software Tool for Complex Proteome Analysis.

PubMed

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.

Systems Proteomics for Translational Network Medicine

PubMed Central

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

HTAPP: High-Throughput Autonomous Proteomic Pipeline

PubMed Central

Yu, Kebing; Salomon, Arthur R.

2011-01-01

Recent advances in the speed and sensitivity of mass spectrometers and in analytical methods, the exponential acceleration of computer processing speeds, and the availability of genomic databases from an array of species and protein information databases have led to a deluge of proteomic data. The development of a lab-based automated proteomic software platform for the automated collection, processing, storage, and visualization of expansive proteomic datasets is critically important. The high-throughput autonomous proteomic pipeline (HTAPP) described here is designed from the ground up to provide critically important flexibility for diverse proteomic workflows and to streamline the total analysis of a complex proteomic sample. This tool is comprised of software that controls the acquisition of mass spectral data along with automation of post-acquisition tasks such as peptide quantification, clustered MS/MS spectral database searching, statistical validation, and data exploration within a user-configurable lab-based relational database. The software design of HTAPP focuses on accommodating diverse workflows and providing missing software functionality to a wide range of proteomic researchers to accelerate the extraction of biological meaning from immense proteomic data sets. Although individual software modules in our integrated technology platform may have some similarities to existing tools, the true novelty of the approach described here is in the synergistic and flexible combination of these tools to provide an integrated and efficient analysis of proteomic samples. PMID:20336676

Achievements and perspectives of top-down proteomics.

PubMed

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.

Functional Proteomic Analysis of Human NucleolusD⃞

PubMed Central

Scherl, Alexander; Couté, Yohann; Déon, Catherine; Callé, Aleth; Kindbeiter, Karine; Sanchez, Jean-Charles; Greco, Anna; Hochstrasser, Denis; Diaz, Jean-Jacques

2002-01-01

The notion of a “plurifunctional” nucleolus is now well established. However, molecular mechanisms underlying the biological processes occurring within this nuclear domain remain only partially understood. As a first step in elucidating these mechanisms we have carried out a proteomic analysis to draw up a list of proteins present within nucleoli of HeLa cells. This analysis allowed the identification of 213 different nucleolar proteins. This catalog complements that of the 271 proteins obtained recently by others, giving a total of ∼350 different nucleolar proteins. Functional classification of these proteins allowed outlining several biological processes taking place within nucleoli. Bioinformatic analyses permitted the assignment of hypothetical functions for 43 proteins for which no functional information is available. Notably, a role in ribosome biogenesis was proposed for 31 proteins. More generally, this functional classification reinforces the plurifunctional nature of nucleoli and provides convincing evidence that nucleoli may play a central role in the control of gene expression. Finally, this analysis supports the recent demonstration of a coupling of transcription and translation in higher eukaryotes. PMID:12429849

A comparative intracellular proteomic profiling of Pseudomonas aeruginosa strain ASP-53 grown on pyrene or glucose as sole source of carbon and identification of some key enzymes of pyrene biodegradation pathway.

PubMed

Mukherjee, Ashis K; Bhagowati, Pabitra; Biswa, Bhim Bahadur; Chanda, Abhishek; Kalita, Bhargab

2017-09-07

Pseudomonas aeruginosa strain ASP-53, isolated from a petroleum oil-contaminated soil sample, was found to be an efficient degrader of pyrene. PCR amplification of selected hydrocarbon catabolic genes (alkB gene, which encodes for monooxygenase, and the C12O, C23O, and PAH-RHDα genes encoding for the dioxygenase enzyme) from the genomic DNA of P. aeruginosa strain ASP-53 suggested its hydrocarbon degradation potential. The GC-MS analysis demonstrated 30.1% pyrene degradation by P. aeruginosa strain ASP-53 after 144h of incubation at pH6.5, 37°C. Expressions of 115 and 196 intracellular proteins were unambiguously identified and quantitated by shotgun proteomics analysis when the isolate was grown in medium containing pyrene and glucose, respectively. The pyrene-induced uniquely expressed and up-regulated proteins in P. aeruginosa strain ASP-53 in addition to substrate (pyrene) metabolism are also likely to be associated with different cellular functions for example-related to protein folding (molecular chaperone), stress response, metabolism of carbohydrate, proteins and amino acids, and fatty acids; transport of metabolites, energy generation such as ATP synthesis, electron transport and nitrate assimilation, and other oxidation-reduction reactions. Proteomic analyses identified some important enzymes involved in pyrene degradation by P. aeruginosa ASP-53 which shows that this bacterium follows the salicylate pathway of pyrene degradation. This study is the first report on proteomic analysis of pyrene biodegradation pathway by Pseudomonas aeruginosa, isolated from a petroleum-oil contaminated soil sample. The pathway displays partial similarity with deduced pyrene degradation mechanisms of Mycobacterium vanbaalenii PYR-1. The GC-MS analysis as well as PCR amplification of hydrocarbon catabolic genes substantiated the potency of the bacterium under study to effectively degrade high molecular weight, toxic PAH such as pyrene for its filed scale bioremediation experiments. The proteomics approach (LC-MS/MS analysis) identified the differentially regulated intracellular proteins of the isolate P. aeruginosa ASP-53 when grown in pyrene medium. This study identified some important pyrene biodegradation enzymes in Pseudomonas aeruginosa ASP-53 and highlights that the bacterium follows salicylate pathway for pyrene degradation. Copyright © 2017 Elsevier B.V. All rights reserved.

Toward the Standardization of Mitochondrial Proteomics: The Italian Mitochondrial Human Proteome Project Initiative.

PubMed

Alberio, Tiziana; Pieroni, Luisa; Ronci, Maurizio; Banfi, Cristina; Bongarzone, Italia; Bottoni, Patrizia; Brioschi, Maura; Caterino, Marianna; Chinello, Clizia; Cormio, Antonella; Cozzolino, Flora; Cunsolo, Vincenzo; Fontana, Simona; Garavaglia, Barbara; Giusti, Laura; Greco, Viviana; Lucacchini, Antonio; Maffioli, Elisa; Magni, Fulvio; Monteleone, Francesca; Monti, Maria; Monti, Valentina; Musicco, Clara; Petrosillo, Giuseppe; Porcelli, Vito; Saletti, Rosaria; Scatena, Roberto; Soggiu, Alessio; Tedeschi, Gabriella; Zilocchi, Mara; Roncada, Paola; Urbani, Andrea; Fasano, Mauro

2017-12-01

The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.

Targeted proteomics identifies liquid-biopsy signatures for extracapsular prostate cancer

PubMed Central

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

Proteomic profile of the Bradysia odoriphaga in response to the microbial secondary metabolite benzothiazole.

PubMed

Zhao, Yunhe; Cui, Kaidi; Xu, Chunmei; Wang, Qiuhong; Wang, Yao; Zhang, Zhengqun; Liu, Feng; Mu, Wei

2016-11-24

Benzothiazole, a microbial secondary metabolite, has been demonstrated to possess fumigant activity against Sclerotinia sclerotiorum, Ditylenchus destructor and Bradysia odoriphaga. However, to facilitate the development of novel microbial pesticides, the mode of action of benzothiazole needs to be elucidated. Here, we employed iTRAQ-based quantitative proteomics analysis to investigate the effects of benzothiazole on the proteomic expression of B. odoriphaga. In response to benzothiazole, 92 of 863 identified proteins in B. odoriphaga exhibited altered levels of expression, among which 14 proteins were related to the action mechanism of benzothiazole, 11 proteins were involved in stress responses, and 67 proteins were associated with the adaptation of B. odoriphaga to benzothiazole. Further bioinformatics analysis indicated that the reduction in energy metabolism, inhibition of the detoxification process and interference with DNA and RNA synthesis were potentially associated with the mode of action of benzothiazole. The myosin heavy chain, succinyl-CoA synthetase and Ca + -transporting ATPase proteins may be related to the stress response. Increased expression of proteins involved in carbohydrate metabolism, energy production and conversion pathways was responsible for the adaptive response of B. odoriphaga. The results of this study provide novel insight into the molecular mechanisms of benzothiazole at a large-scale translation level and will facilitate the elucidation of the mechanism of action of benzothiazole.

Polarity-based fractionation in proteomics: hydrophilic interaction vs reversed-phase liquid chromatography.

PubMed

Jafari, M; Mirzaie, M; Khodabandeh, M; Rezadoost, H; Ghassempour, A; Aboul-Enein, H Y

2016-07-01

During recent decades, hydrophilic interaction liquid chromatography (HILIC) ahs been introduced to fractionate or purify especially polar solutes such as peptides and proteins while reversed-phase liquid chromatography (RPLC) is also a common strategy. RPLC is also a common dimension in multidimensional chromatography. In this study, the potential of HILIC vs RPLC chromatography was compared for proteome mapping of human peripheral blood mononuclear cell extract. In HILIC a silica-based stationary phase and for RPLC a C18 column were applied. Then separated proteins were eluted to an ion trap mass spectrometry system. Our results showed that the HILIC leads to more proteins being identified in comparison to RPLC. Among the total 181 identified proteins, 56 and 38 proteins were fractionated specifically by HILIC and RPLC, respectively. In order to demonstrate this, the physicochemical properties of identified proteins such as polarity and hydrophobicity were considered. This analysis indicated that polarity may play a major role in the HILIC separation of proteins vs RPLC. Using gene ontology enrichment analysis, it was also observed that differences in physicochemical properties conform to the cellular compartment and biological features. Finally, this study highlighted the potential of HILIC and the great orthogonality of RPLC in gel-free proteomic studies. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Hydrophilic Strong Anion Exchange (hSAX) Chromatography for Highly Orthogonal Peptide Separation of Complex Proteomes

PubMed Central

2013-01-01

Due to its compatibility and orthogonality to reversed phase (RP) liquid chromatography (LC) separation, ion exchange chromatography, and mainly strong cation exchange (SCX), has often been the first choice in multidimensional LC experiments in proteomics. Here, we have tested the ability of three strong anion exchanger (SAX) columns differing in their hydrophobicity to fractionate RAW264.7 macrophage cell lysate. IonPac AS24, a strong anion exchange material with ultralow hydrophobicity, demonstrated to be superior to other materials by fractionation and separation of tryptic peptides from both a mixture of 6 proteins as well as mouse cell lysate. The chromatography displayed very high orthogonality and high robustness depending on the hydrophilicity of column chemistry, which we termed hydrophilic strong anion exchange (hSAX). Mass spectrometry analysis of 34 SAX fractions from RAW264.7 macrophage cell lysate digest resulted in an identification of 9469 unique proteins and 126318 distinct peptides in one week of instrument time. Moreover, when compared to an optimized high pH/low pH RP separation approach, the method presented here raised the identification of proteins and peptides by 10 and 28%, respectively. This novel hSAX approach provides robust, reproducible, and highly orthogonal separation of complex protein digest samples for deep coverage proteome analysis. PMID:23294059

Proteogenomics connects somatic mutations to signalling in breast cancer.

PubMed

Mertins, Philipp; Mani, D R; Ruggles, Kelly V; Gillette, Michael A; Clauser, Karl R; Wang, Pei; Wang, Xianlong; Qiao, Jana W; Cao, Song; Petralia, Francesca; Kawaler, Emily; Mundt, Filip; Krug, Karsten; Tu, Zhidong; Lei, Jonathan T; Gatza, Michael L; Wilkerson, Matthew; Perou, Charles M; Yellapantula, Venkata; Huang, Kuan-lin; Lin, Chenwei; McLellan, Michael D; Yan, Ping; Davies, Sherri R; Townsend, R Reid; Skates, Steven J; Wang, Jing; Zhang, Bing; Kinsinger, Christopher R; Mesri, Mehdi; Rodriguez, Henry; Ding, Li; Paulovich, Amanda G; Fenyö, David; Ellis, Matthew J; Carr, Steven A

2016-06-02

Somatic mutations have been extensively characterized in breast cancer, but the effects of these genetic alterations on the proteomic landscape remain poorly understood. Here we describe quantitative mass-spectrometry-based proteomic and phosphoproteomic analyses of 105 genomically annotated breast cancers, of which 77 provided high-quality data. Integrated analyses provided insights into the somatic cancer genome including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. Interrogation of the 5q trans-effects against the Library of Integrated Network-based Cellular Signatures, connected loss of CETN3 and SKP1 to elevated expression of epidermal growth factor receptor (EGFR), and SKP1 loss also to increased SRC tyrosine kinase. Global proteomic data confirmed a stromal-enriched group of proteins in addition to basal and luminal clusters, and pathway analysis of the phosphoproteome identified a G-protein-coupled receptor cluster that was not readily identified at the mRNA level. In addition to ERBB2, other amplicon-associated highly phosphorylated kinases were identified, including CDK12, PAK1, PTK2, RIPK2 and TLK2. We demonstrate that proteogenomic analysis of breast cancer elucidates the functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies therapeutic targets.

An introduction to statistical process control in research proteomics.

PubMed

Bramwell, David

2013-12-16

Statistical process control is a well-established and respected method which provides a general purpose, and consistent framework for monitoring and improving the quality of a process. It is routinely used in many industries where the quality of final products is critical and is often required in clinical diagnostic laboratories [1,2]. To date, the methodology has been little utilised in research proteomics. It has been shown to be capable of delivering quantitative QC procedures for qualitative clinical assays [3] making it an ideal methodology to apply to this area of biological research. To introduce statistical process control as an objective strategy for quality control and show how it could be used to benefit proteomics researchers and enhance the quality of the results they generate. We demonstrate that rules which provide basic quality control are easy to derive and implement and could have a major impact on data quality for many studies. Statistical process control is a powerful tool for investigating and improving proteomics research work-flows. The process of characterising measurement systems and defining control rules forces the exploration of key questions that can lead to significant improvements in performance. This work asserts that QC is essential to proteomics discovery experiments. Every experimenter must know the current capabilities of their measurement system and have an objective means for tracking and ensuring that performance. Proteomic analysis work-flows are complicated and multi-variate. QC is critical for clinical chemistry measurements and huge strides have been made in ensuring the quality and validity of results in clinical biochemistry labs. This work introduces some of these QC concepts and works to bridge their use from single analyte QC to applications in multi-analyte systems. This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics. Copyright © 2013 The Author. Published by Elsevier B.V. All rights reserved.

Simple preparation of plant epidermal tissue for laser microdissection and downstream quantitative proteome and carbohydrate analysis

PubMed Central

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

Simple preparation of plant epidermal tissue for laser microdissection and downstream quantitative proteome and carbohydrate analysis.

PubMed

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.

Proteomic Identification of Heat Shock-Induced Danger Signals in a Melanoma Cell Lysate Used in Dendritic Cell-Based Cancer Immunotherapy.

PubMed

González, Fermín E; Chernobrovkin, Alexey; Pereda, Cristián; García-Salum, Tamara; Tittarelli, Andrés; López, Mercedes N; Salazar-Onfray, Flavio; Zubarev, Roman A

2018-01-01

Autologous dendritic cells (DCs) loaded with cancer cell-derived lysates have become a promising tool in cancer immunotherapy. During the last decade, we demonstrated that vaccination of advanced melanoma patients with autologous tumor antigen presenting cells (TAPCells) loaded with an allogeneic heat shock- (HS-) conditioned melanoma cell-derived lysate (called TRIMEL) is able to induce an antitumor immune response associated with a prolonged patient survival. TRIMEL provides not only a broad spectrum of potential melanoma-associated antigens but also danger signals that are crucial in the induction of a committed mature DC phenotype. However, potential changes induced by heat conditioning on the proteome of TRIMEL are still unknown. The identification of newly or differentially expressed proteins under defined stress conditions is relevant for understanding the lysate immunogenicity. Here, we characterized the proteomic profile of TRIMEL in response to HS treatment. A quantitative label-free proteome analysis of over 2800 proteins was performed, with 91 proteins that were found to be regulated by HS treatment: 18 proteins were overexpressed and 73 underexpressed. Additionally, 32 proteins were only identified in the HS-treated TRIMEL and 26 in non HS-conditioned samples. One protein from the overexpressed group and two proteins from the HS-exclusive group were previously described as potential damage-associated molecular patterns (DAMPs). Some of the HS-induced proteins, such as haptoglobin, could be also considered as DAMPs and candidates for further immunological analysis in the establishment of new putative danger signals with immunostimulatory functions.

Proteomic Identification of Heat Shock-Induced Danger Signals in a Melanoma Cell Lysate Used in Dendritic Cell-Based Cancer Immunotherapy

PubMed Central

Chernobrovkin, Alexey; Pereda, Cristián; García-Salum, Tamara; Tittarelli, Andrés; López, Mercedes N.; Salazar-Onfray, Flavio

2018-01-01

Autologous dendritic cells (DCs) loaded with cancer cell-derived lysates have become a promising tool in cancer immunotherapy. During the last decade, we demonstrated that vaccination of advanced melanoma patients with autologous tumor antigen presenting cells (TAPCells) loaded with an allogeneic heat shock- (HS-) conditioned melanoma cell-derived lysate (called TRIMEL) is able to induce an antitumor immune response associated with a prolonged patient survival. TRIMEL provides not only a broad spectrum of potential melanoma-associated antigens but also danger signals that are crucial in the induction of a committed mature DC phenotype. However, potential changes induced by heat conditioning on the proteome of TRIMEL are still unknown. The identification of newly or differentially expressed proteins under defined stress conditions is relevant for understanding the lysate immunogenicity. Here, we characterized the proteomic profile of TRIMEL in response to HS treatment. A quantitative label-free proteome analysis of over 2800 proteins was performed, with 91 proteins that were found to be regulated by HS treatment: 18 proteins were overexpressed and 73 underexpressed. Additionally, 32 proteins were only identified in the HS-treated TRIMEL and 26 in non HS-conditioned samples. One protein from the overexpressed group and two proteins from the HS-exclusive group were previously described as potential damage-associated molecular patterns (DAMPs). Some of the HS-induced proteins, such as haptoglobin, could be also considered as DAMPs and candidates for further immunological analysis in the establishment of new putative danger signals with immunostimulatory functions. PMID:29744371

Proteomic analysis of corneal endothelial cell-descemet membrane tissues reveals influence of insulin dependence and disease severity in type 2 diabetes mellitus.

PubMed

Skeie, Jessica M; Aldrich, Benjamin T; Goldstein, Andrew S; Schmidt, Gregory A; Reed, Cynthia R; Greiner, Mark A

2018-01-01

The objective of this study was to characterize the proteome of the corneal endothelial cell layer and its basement membrane (Descemet membrane) in humans with various severities of type II diabetes mellitus compared to controls, and identify differentially expressed proteins across a range of diabetic disease severities that may influence corneal endothelial cell health. Endothelium-Descemet membrane complex tissues were peeled from transplant suitable donor corneas. Protein fractions were isolated from each sample and subjected to multidimensional liquid chromatography and tandem mass spectrometry. Peptide spectra were matched to the human proteome, assigned gene ontology, and grouped into protein signaling pathways unique to each of the disease states. We identified an average of 12,472 unique proteins in each of the endothelium-Descemet membrane complex tissue samples. There were 2,409 differentially expressed protein isoforms that included previously known risk factors for type II diabetes mellitus related to metabolic processes, oxidative stress, and inflammation. Gene ontology analysis demonstrated that diabetes progression has many protein footprints related to metabolic processes, binding, and catalysis. The most represented pathways involved in diabetes progression included mitochondrial dysfunction, cell-cell junction structure, and protein synthesis regulation. This proteomic dataset identifies novel corneal endothelial cell and Descemet membrane protein expression in various stages of diabetic disease. These findings give insight into the mechanisms involved in diabetes progression relevant to the corneal endothelium and its basement membrane, prioritize new pathways for therapeutic targeting, and provide insight into potential biomarkers for determining the health of this tissue.

Multidimensional proteomics for cell biology.

PubMed

Larance, Mark; Lamond, Angus I

2015-05-01

The proteome is a dynamic system in which each protein has interconnected properties - dimensions - that together contribute to the phenotype of a cell. Measuring these properties has proved challenging owing to their diversity and dynamic nature. Advances in mass spectrometry-based proteomics now enable the measurement of multiple properties for thousands of proteins, including their abundance, isoform expression, turnover rate, subcellular localization, post-translational modifications and interactions. Complementing these experimental developments are new data analysis, integration and visualization tools as well as data-sharing resources. Together, these advances in the multidimensional analysis of the proteome are transforming our understanding of various cellular and physiological processes.

CPTAC Releases Cancer Proteome Confirmatory Ovarian Study Data | Office of Cancer Clinical Proteomics Research

Cancer.gov

A catalogue of molecular aberrations that cause ovarian cancer is critical for developing and deploying diagnostics and therapies that will improve patients’ lives. Because a comprehensive molecular view of cancer is important for ultimately guiding treatment, the National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) has released the cancer proteome confirmatory ovarian study data sets.

Advances of Proteomic Sciences in Dentistry.

PubMed

Khurshid, Zohaib; Zohaib, Sana; Najeeb, Shariq; Zafar, Muhammad Sohail; Rehman, Rabia; Rehman, Ihtesham Ur

2016-05-13

Applications of proteomics tools revolutionized various biomedical disciplines such as genetics, molecular biology, medicine, and dentistry. The aim of this review is to highlight the major milestones in proteomics in dentistry during the last fifteen years. Human oral cavity contains hard and soft tissues and various biofluids including saliva and crevicular fluid. Proteomics has brought revolution in dentistry by helping in the early diagnosis of various diseases identified by the detection of numerous biomarkers present in the oral fluids. This paper covers the role of proteomics tools for the analysis of oral tissues. In addition, dental materials proteomics and their future directions are discussed.

Halobacterium salinarum NRC-1 PeptideAtlas: toward strategies for targeted proteomics and improved proteome coverage.

PubMed

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.

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

PubMed

Trugilho, Monique Ramos de Oliveira; Hottz, Eugenio Damaceno; Brunoro, Giselle Villa Flor; Teixeira-Ferreira, André; Carvalho, Paulo Costa; Salazar, Gustavo Adolfo; Zimmerman, Guy A; Bozza, Fernando A; Bozza, Patrícia T; Perales, Jonas

2017-05-01

Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses.

Long-Gradient Separations Coupled with Selected Reaction Monitoring for Highly Sensitive, Large Scale Targeted Protein Quantification in a Single Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Fillmore, Thomas L.; Gao, Yuqian

2013-10-01

Long-gradient separations coupled to tandem MS were recently demonstrated to provide a deep proteome coverage for global proteomics; however, such long-gradient separations have not been explored for targeted proteomics. Herein, we investigate the potential performance of the long-gradient separations coupled with selected reaction monitoring (LG-SRM) for targeted protein quantification. Direct comparison of LG-SRM (5 h gradient) and conventional LC-SRM (45 min gradient) showed that the long-gradient separations significantly reduced background interference levels and provided an 8- to 100-fold improvement in LOQ for target proteins in human female serum. Based on at least one surrogate peptide per protein, an LOQ ofmore » 10 ng/mL was achieved for the two spiked proteins in non-depleted human serum. The LG-SRM detection of seven out of eight endogenous plasma proteins expressed at ng/mL or sub-ng/mL levels in clinical patient sera was also demonstrated. A correlation coefficient of >0.99 was observed for the results of LG-SRM and ELISA measurements for prostate-specific antigen (PSA) in selected patient sera. Further enhancement of LG-SRM sensitivity was achieved by applying front-end IgY14 immunoaffinity depletion. Besides improved sensitivity, LG-SRM offers at least 3 times higher multiplexing capacity than conventional LC-SRM due to ~3-fold increase in average peak widths for a 300-min gradient compared to a 45-min gradient. Therefore, LG-SRM holds great potential for bridging the gap between global and targeted proteomics due to its advantages in both sensitivity and multiplexing capacity.« less

Characterization and Comprehensive Proteome Profiling of Exosomes Secreted by Hepatocytes

PubMed Central

Conde-Vancells, Javier; Rodriguez-Suarez, Eva; Embade, Nieves; Gil, David; Matthiesen, Rune; Valle, Mikel; Elortza, Felix; Lu, Shelly C.; Mato, Jose M.; Falcon-Perez, Juan M.

2009-01-01

Synopsis Exosomes constitute a discrete population of nanometer-sized (30-150 nm) vesicles formed in endocytic compartments and released to the extracellular environment by different cell types. In this work we demonstrated by electron microscopic, western blotting and proteomic analyses that primary hepatocytes secrete exosome-like vesicles containing proteins involved in metabolizing lipoproteins, endogenous compounds as well as xenobiotics. These new findings contribute to improve our knowledge about biology's hepatocyte and may have important diagnostic, prognosis and therapeutic implications in liver diseases Exosomes represent a discrete population of vesicles that are secreted from various cell types to the extracellular media. Their protein and lipid composition are a consequence of sorting events at the level of the multivesicular body, a central organelle which integrates endocytic and secretory pathways. Characterization of exosomes from different biological samples has shown the presence of common as well as cell-type specific proteins. Remarkably, the protein content of the exosomes is modified upon pathological or stress conditions. Hepatocytes play a central role in the body response to stress metabolizing potentially harmful endogenous substances as well as xenobiotics. In the present study we described and characterized for first time exosome secretion in non-tumoral hepatocytes, and using a systematic proteomic approach, we establish the first extensive proteome of a hepatocyte-derived exosome population which should be useful in furthering our understanding of the hepatic function and in the identification of components that may serve as biomarkers for hepatic alterations. Our analysis identifies a significant number of proteins previously described among exosomes derived from others cell types as well as proteins involved in metabolizing lipoproteins, endogenous compounds and xenobiotics, not previously described in exosomes. Furthermore, we demonstrated that exosomal membrane proteins can constitute an interesting tool to express non-exosomal proteins into exosomes with therapeutic purposes. PMID:19367702

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue

PubMed Central

Teixeira-Ferreira, André; Carvalho, Paulo Costa; Salazar, Gustavo Adolfo; Zimmerman, Guy A.; Perales, Jonas

2017-01-01

Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses. PMID:28542641

Identifying Urinary and Serum Exosome Biomarkers for Radiation Exposure Using a Data Dependent Acquisition and SWATH-MS Combined Workflow.

PubMed

Kulkarni, Shilpa; Koller, Antonius; Mani, Kartik M; Wen, Ruofeng; Alfieri, Alan; Saha, Subhrajit; Wang, Jian; Patel, Purvi; Bandeira, Nuno; Guha, Chandan; Chen, Emily I

2016-11-01

Early and accurate assessment of radiation injury by radiation-responsive biomarkers is critical for triage and early intervention. Biofluids such as urine and serum are convenient for such analysis. Recent research has also suggested that exosomes are a reliable source of biomarkers in disease progression. In the present study, we analyzed total urine proteome and exosomes isolated from urine or serum for potential biomarkers of acute and persistent radiation injury in mice exposed to lethal whole body irradiation (WBI). For feasibility studies, the mice were irradiated at 10.4 Gy WBI, and urine and serum samples were collected 24 and 72 hours after irradiation. Exosomes were isolated and analyzed using liquid chromatography mass spectrometry/mass spectrometry-based workflow for radiation exposure signatures. A data dependent acquisition and SWATH-MS combined workflow approach was used to identify significantly exosome biomarkers indicative of acute or persistent radiation-induced responses. For the validation studies, mice were exposed to 3, 6, 8, or 10 Gy WBI, and samples were analyzed for comparison. A comparison between total urine proteomics and urine exosome proteomics demonstrated that exosome proteomic analysis was superior in identifying radiation signatures. Feasibility studies identified 23 biomarkers from urine and 24 biomarkers from serum exosomes after WBI. Urinary exosome signatures identified different physiological parameters than the ones obtained in serum exosomes. Exosome signatures from urine indicated injury to the liver, gastrointestinal, and genitourinary tracts. In contrast, serum showed vascular injuries and acute inflammation in response to radiation. Selected urinary exosomal biomarkers also showed changes at lower radiation doses in validation studies. Exosome proteomics revealed radiation- and time-dependent protein signatures after WBI. A total of 47 differentially secreted proteins were identified in urinary and serum exosomes. Together, these data showed the feasibility of defining biomarkers that could elucidate tissue-associated and systemic response caused by high-dose ionizing radiation. This is the first report using an exosome proteomics approach to identify radiation signatures. Copyright © 2016 Elsevier Inc. All rights reserved.

Identifying Urinary and Serum Exosome Biomarkers for Radiation Exposure Using a Data Dependent Acquisition and SWATH-MS Combined Workflow

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kulkarni, Shilpa; Koller, Antonius; Proteomics Shared Resource, Herbert Irving Comprehensive Cancer Center, New York, New York

Purpose: Early and accurate assessment of radiation injury by radiation-responsive biomarkers is critical for triage and early intervention. Biofluids such as urine and serum are convenient for such analysis. Recent research has also suggested that exosomes are a reliable source of biomarkers in disease progression. In the present study, we analyzed total urine proteome and exosomes isolated from urine or serum for potential biomarkers of acute and persistent radiation injury in mice exposed to lethal whole body irradiation (WBI). Methods and Materials: For feasibility studies, the mice were irradiated at 10.4 Gy WBI, and urine and serum samples were collected 24more » and 72 hours after irradiation. Exosomes were isolated and analyzed using liquid chromatography mass spectrometry/mass spectrometry-based workflow for radiation exposure signatures. A data dependent acquisition and SWATH-MS combined workflow approach was used to identify significantly exosome biomarkers indicative of acute or persistent radiation-induced responses. For the validation studies, mice were exposed to 3, 6, 8, or 10 Gy WBI, and samples were analyzed for comparison. Results: A comparison between total urine proteomics and urine exosome proteomics demonstrated that exosome proteomic analysis was superior in identifying radiation signatures. Feasibility studies identified 23 biomarkers from urine and 24 biomarkers from serum exosomes after WBI. Urinary exosome signatures identified different physiological parameters than the ones obtained in serum exosomes. Exosome signatures from urine indicated injury to the liver, gastrointestinal, and genitourinary tracts. In contrast, serum showed vascular injuries and acute inflammation in response to radiation. Selected urinary exosomal biomarkers also showed changes at lower radiation doses in validation studies. Conclusions: Exosome proteomics revealed radiation- and time-dependent protein signatures after WBI. A total of 47 differentially secreted proteins were identified in urinary and serum exosomes. Together, these data showed the feasibility of defining biomarkers that could elucidate tissue-associated and systemic response caused by high-dose ionizing radiation. This is the first report using an exosome proteomics approach to identify radiation signatures.« less

High-resolution ultrahigh-pressure long column reversed-phase liquid chromatography for top-down proteomics.

PubMed

Shen, Yufeng; Tolić, Nikola; Piehowski, Paul D; Shukla, Anil K; Kim, Sangtae; Zhao, Rui; Qu, Yi; Robinson, Errol; Smith, Richard D; Paša-Tolić, Ljiljana

2017-05-19

Separation of proteoforms for global intact protein analysis (i.e. top-down proteomics) has lagged well behind what is achievable for peptides in traditional bottom-up proteomic approach and is becoming a true bottle neck for top-down proteomics. Herein, we report use of long (≥1M) columns containing short alkyl (C1-C4) bonded phases to achieve high-resolution RPLC for separation of proteoforms. At a specific operation pressure limit (i.e., 96.5MPa or 14Kpsi used in this work), column length was found to be the most important factor for achieving maximal resolution separation of proteins when 1.5-5μm particles were used as packings and long columns provided peak capacities greater than 400 for proteoforms derived from a global cell lysate with molecular weights below 50kDa. Larger proteoforms (50-110kDa) were chromatographed on long RPLC columns and detected by MS; however, they cannot be identified yet by tandem mass spectrometry. Our experimental data further demonstrated that long alkyl (e.g., C8 and C18) bonded particles provided high-resolution RPLC for <10kDa proteoforms, not efficient for separation of global proteoforms. Reversed-phase particles with porous, nonporous, and superficially porous surfaces were systematically investigated for high-resolution RPLC. Pore size (200-400Å) and the surface structure (porous and superficially porous) of particles was found to have minor influences on high-resolution RPLC of proteoforms. RPLC presented herein enabled confident identification of ∼900 proteoforms (1% FDR) for a low-microgram quantity of proteomic samples using a single RPLC-MS/MS analysis. The level of RPLC performance attained in this work is close to that typically realized in bottom-up proteomics, and broadly useful when applying e.g., the single-stage MS accurate mass tag approach, but less effective when combined with current tandem MS. Our initial data indicate that MS detection and fragmentation inefficiencies provided by current high-resolution mass spectrometers are key challenges for characterization of larger proteoforms. Copyright © 2017. Published by Elsevier B.V.

High-resolution ultrahigh-pressure long column reversed-phase liquid chromatography for top-down proteomics

DOE PAGES

Shen, Yufeng; Tolić, Nikola; Piehowski, Paul D.; ...

2017-01-05

Separation of proteoforms for global intact protein analysis (i.e. top-down proteomics) has lagged well behind what is achievable for peptides in traditional bottom-up proteomic approach and is becoming a true bottle neck for top-down proteomics. We report use of long (≥1 M) columns containing short alkyl (C1-C4) bonded phases to achieve high-resolution RPLC for separation of proteoforms. At a specific operation pressure limit (i.e., 96.5 MPa or 14 K psi used in this work), column length was found to be the most important factor for achieving maximal resolution separation of proteins when 1.5–5 μm particles were used as packings andmore » long columns provided peak capacities greater than 400 for proteoforms derived from a global cell lysate with molecular weights below 50 kDa. Furthermore, we chromatographed larger proteoforms (50–110 kDa) on long RPLC columns and detected by MS; however, they cannot be identified yet by tandem mass spectrometry. Our experimental data further demonstrated that long alkyl (e.g., C8 and C18) bonded particles provided high-resolution RPLC for <10 kDa proteoforms, not efficient for separation of global proteoforms. Reversed-phase particles with porous, nonporous, and superficially porous surfaces were systematically investigated for high-resolution RPLC. Pore size (200–400 Å) and the surface structure (porous and superficially porous) of particles was found to have minor influences on high-resolution RPLC of proteoforms. RPLC presented herein enabled confident identification of ~900 proteoforms (1% FDR) for a low-microgram quantity of proteomic samples using a single RPLC–MS/MS analysis. The level of RPLC performance attained in this work is close to that typically realized in bottom-up proteomics, and broadly useful when applying e.g., the single-stage MS accurate mass tag approach, but less effective when combined with current tandem MS. Finally, our initial data indicate that MS detection and fragmentation inefficiencies provided by current high-resolution mass spectrometers are key challenges for characterization of larger proteoforms.« less

ISSLS PRIZE IN CLINICAL SCIENCE 2017: Is infection the possible initiator of disc disease? An insight from proteomic analysis.

PubMed

Rajasekaran, S; Tangavel, Chitraa; Aiyer, Siddharth N; Nayagam, Sharon Miracle; Raveendran, M; Demonte, Naveen Luke; Subbaiah, Pramela; Kanna, Rishi; Shetty, Ajoy Prasad; Dharmalingam, K

2017-05-01

Proteomic and 16S rDNA analysis of disc tissues obtained in vivo. To address the controversy of infection as an aetiology for disc disorders through protein profiling. There is raging controversy over the presence of bacteria in human lumbar discs in vivo, and if they represent contamination or infection. Proteomics can provide valuable insight by identifying proteins signifying bacterial presence and, also host defence response proteins (HDRPs), which will confirm infection. 22 discs (15-disc herniations (DH), 5-degenerate (DD), 2-normal in MRI (NM) were harvested intraoperatively and immediately snap frozen. Samples were pooled into three groups and proteins extracted were analysed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Post identification, data analysis was performed using Uniprotdb, Pantherdb, Proteome discoverer and STRING network. Authentication for bacterial presence was performed by PCR amplification of 16S rDNA. LC-MS/MS analysis using Orbitrap showed 1103 proteins in DH group, compared to 394 in NM and 564 in DD. 73 bacterial specific proteins were identified (56 specific for Propionibacterium acnes; 17 for Staphylococcus epidermidis). In addition, 67 infection-specific HDRPs, unique or upregulated, such as Defensin, Lysozyme, Dermcidin, Cathepsin-G, Prolactin-Induced Protein, and Phospholipase-A2, were identified confirming presence of infection. Species-specific primers for P. acnes exhibited amplicons at 946 bp (16S rDNA) and 515 bp (Lipase) confirming presence of P. acnes in both NM discs, 11 of 15 DH discs, and all five DD discs. Bioinformatic search for protein-protein interactions (STRING) documented 169 proteins with close interactions (protein clustering co-efficient 0.7) between host response and degenerative proteins implying that infection may initiate degradation through Ubiquitin C. Our study demonstrates bacterial specific proteins and host defence proteins to infection which strengthen the hypothesis of infection as a possible initiator of disc disease. These results can lead to a paradigm shift in our understanding and management of disc disorders.

Hormone regulation of rhizome development in tall fescue (Festuca arundinacea) associated with proteomic changes controlling respiratory and amino acid metabolism.

PubMed

Ma, Xiqing; Xu, Qian; Meyer, William A; Huang, Bingru

2016-09-01

Rhizomes are underground stems with meristematic tissues capable of generating shoots and roots. However, mechanisms controlling rhizome formation and growth are yet to be completely understood. The objectives of this study were to investigate whether rhizome development could be regulated by cytokinins (CKs) and gibberellic acids (GAs), and determine underlying mechanisms of regulation of rhizome formation and growth of tall fescue (Festuca arundinacea) by a CK or GA through proteomic and transcript analysis. A rhizomatous genotype of tall fescue ('BR') plants were treated with 6-benzylaminopurine (BAP, a synthetic cytokinin) or GA3 in hydroponic culture in growth chambers. Furthermore, comparative proteomic analysis of two-dimensional electrophoresis and mass spectrometry were performed to investigate proteins and associated metabolic pathways imparting increased rhizome number by BAP and rhizome elongation by GA3 KEY RESULTS: BAP stimulated rhizome formation while GA3 promoted rhizome elongation. Proteomic analysis identified 76 differentially expressed proteins (DEPs) due to BAP treatment and 37 DEPs due to GA3 treatment. Cytokinin-related genes and cell division-related genes were upregulated in the rhizome node by BAP and gibberellin-related and cell growth-related genes in the rhizome by GA3 CONCLUSIONS: Most of the BAP- or GA-responsive DEPs were involved in respiratory metabolism and amino acid metabolism. Transcription analysis demonstrated that genes involved in hormone metabolism, signalling pathways, cell division and cell-wall loosening were upregulated by BAP or GA3 The CK and GA promoted rhizome formation and growth, respectively, by activating metabolic pathways that supply energy and amino acids to support cell division and expansion during rhizome initiation and elongation in tall fescue. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

CPTAC Collaborates with Molecular & Cellular Proteomics to Address Reproducibility in Targeted Assay Development | Office of Cancer Clinical Proteomics Research

Cancer.gov

The journal Molecular & Cellular Proteomics (MCP), in collaboration with the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI), part of the National Institutes of Health, announce new guidelines and requirements for papers describing the development and application of targeted mass spectrometry measurements of peptides, modified peptides and proteins (Mol Cell Proteomics 2017; PMID: 28183812).  NCI’s participation is part of NIH’s overall effort to address the r

CPTAC Releases Cancer Proteome Confirmatory Breast Study Data | Office of Cancer Clinical Proteomics Research

Cancer.gov

An estimated 252,710 new cases of female breast cancer, accounting for 15% of all new cancer cases, occurred in 2017. To better understand proteogenomic abnormalities in breast cancer, the National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) announces the release of the cancer proteome confirmatory breast study data. The goal of the study was to comprehensively characterize the proteome and phosphoproteome on approximately 100 prospectively collected breast tumor and adjacent normal tissues.

Proteomic analysis of bovine nucleolus.

PubMed

Patel, Amrutlal K; Olson, Doug; Tikoo, Suresh K

2010-09-01

Nucleolus is the most prominent subnuclear structure, which performs a wide variety of functions in the eukaryotic cellular processes. In order to understand the structural and functional role of the nucleoli in bovine cells, we analyzed the proteomic composition of the bovine nucleoli. The nucleoli were isolated from Madin Darby bovine kidney cells and subjected to proteomic analysis by LC-MS/MS after fractionation by SDS-PAGE and strong cation exchange chromatography. Analysis of the data using the Mascot database search and the GPM database search identified 311 proteins in the bovine nucleoli, which contained 22 proteins previously not identified in the proteomic analysis of human nucleoli. Analysis of the identified proteins using the GoMiner software suggested that the bovine nucleoli contained proteins involved in ribosomal biogenesis, cell cycle control, transcriptional, translational and post-translational regulation, transport, and structural organization. Copyright © 2010 Beijing Genomics Institute. Published by Elsevier Ltd. All rights reserved.

Genetic and biochemical characterization of the GH72 family of cell wall transglycosylases in Neurospora crassa.

PubMed

Ao, Jie; Free, Stephen J

2017-04-01

The Neurospora crassa genome encodes five GH72 family transglycosylases, and four of these enzymes (GEL-1, GEL-2, GEL-3 and GEL-5) have been found to be present in the cell wall proteome. We carried out an extensive genetic analysis on the role of these four transglycosylases in cell wall biogenesis and demonstrated that the transglycosylases are required for the formation of a normal cell wall. As suggested by the proteomic analysis, we found that multiple transglycosylases were being expressed in N. crassa cells and that different combinations of the enzymes are required in different cell types. The combination of GEL-1, GEL-2 and GEL-5 is required for the growth of vegetative hyphae, while the GEL-1, GEL-2, GEL-3 combination is needed for the production of aerial hyphae and conidia. Our data demonstrates that the enzymes are redundant with partially overlapping enzymatic activities, which provides the fungus with a robust cell wall biosynthetic system. Characterization of the transglycosylase-deficient mutants demonstrated that the incorporation of cell wall proteins was severely compromised. Interestingly, we found that the transglycosylase-deficient mutant cell walls contained more β-1,3-glucan than the wild type cell wall. Our results demonstrate that the GH72 transglycosylases are not needed for the incorporation of β-1,3-glucan into the cell wall, but they are required for the incorporation of cell wall glycoprotein into the cell wall. Copyright © 2017 Elsevier Inc. All rights reserved.

YPED: An Integrated Bioinformatics Suite and Database for Mass Spectrometry-based Proteomics Research

PubMed Central

Colangelo, Christopher M.; Shifman, Mark; Cheung, Kei-Hoi; Stone, Kathryn L.; Carriero, Nicholas J.; Gulcicek, Erol E.; Lam, TuKiet T.; Wu, Terence; Bjornson, Robert D.; Bruce, Can; Nairn, Angus C.; Rinehart, Jesse; Miller, Perry L.; Williams, Kenneth R.

2015-01-01

We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database (YPED) that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a single laboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography–tandem mass spectrometry (LC–MS/MS) database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring (MRM)/selective reaction monitoring (SRM) assay development. We have linked YPED’s database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results. PMID:25712262

QPROT: Statistical method for testing differential expression using protein-level intensity data in label-free quantitative proteomics.

PubMed

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.

YPED: an integrated bioinformatics suite and database for mass spectrometry-based proteomics research.

PubMed

Colangelo, Christopher M; Shifman, Mark; Cheung, Kei-Hoi; Stone, Kathryn L; Carriero, Nicholas J; Gulcicek, Erol E; Lam, TuKiet T; Wu, Terence; Bjornson, Robert D; Bruce, Can; Nairn, Angus C; Rinehart, Jesse; Miller, Perry L; Williams, Kenneth R

2015-02-01

We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database (YPED) that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a single laboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography-tandem mass spectrometry (LC-MS/MS) database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring (MRM)/selective reaction monitoring (SRM) assay development. We have linked YPED's database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: Reproducibility, linearity, and application with complex proteomes.

PubMed

Wang, Guanghui; Wu, Wells W; Zeng, Weihua; Chou, Chung-Lin; Shen, Rong-Fong

2006-05-01

A critical step in protein biomarker discovery is the ability to contrast proteomes, a process referred generally as quantitative proteomics. While stable-isotope labeling (e.g., ICAT, 18O- or 15N-labeling, or AQUA) remains the core technology used in mass spectrometry-based proteomic quantification, increasing efforts have been directed to the label-free approach that relies on direct comparison of peptide peak areas between LC-MS runs. This latter approach is attractive to investigators for its simplicity as well as cost effectiveness. In the present study, the reproducibility and linearity of using a label-free approach to highly complex proteomes were evaluated. Various amounts of proteins from different proteomes were subjected to repeated LC-MS analyses using an ion trap or Fourier transform mass spectrometer. Highly reproducible data were obtained between replicated runs, as evidenced by nearly ideal Pearson's correlation coefficients (for ion's peak areas or retention time) and average peak area ratios. In general, more than 50% and nearly 90% of the peptide ion ratios deviated less than 10% and 20%, respectively, from the average in duplicate runs. In addition, the multiplicity ratios of the amounts of proteins used correlated nicely with the observed averaged ratios of peak areas calculated from detected peptides. Furthermore, the removal of abundant proteins from the samples led to an improvement in reproducibility and linearity. A computer program has been written to automate the processing of data sets from experiments with groups of multiple samples for statistical analysis. Algorithms for outlier-resistant mean estimation and for adjusting statistical significance threshold in multiplicity of testing were incorporated to minimize the rate of false positives. The program was applied to quantify changes in proteomes of parental and p53-deficient HCT-116 human cells and found to yield reproducible results. Overall, this study demonstrates an alternative approach that allows global quantification of differentially expressed proteins in complex proteomes. The utility of this method to biomarker discovery is likely to synergize with future improvements in the detecting sensitivity of mass spectrometers.

Mass Spectrometry Data Collection in Parallel at Multiple Core Facilities Operating TripleTOF 5600 and Orbitrap Elite/Velos Pro/Q Exactive Mass Spectrometers

PubMed Central

Jones, K.; Kim, K.; Patel, B.; Kelsen, S.; Braverman, A.; Swinton, D.; Gafken, P.; Jones, L.; Lane, W.; Neveu, J.; Leung, H.; Shaffer, S.; Leszyk, J.; Stanley, B.; Fox, T.; Stanley, A.; Yeung, Anthony

2013-01-01

Proteomic research can benefit from simultaneous access to multiple cutting-edge mass spectrometers. 18 core facilities responded to our investigators seeking service through the ABRF Discussion Forum. Five of the facilities selected completed four plasma proteomics experiments as routine fee-for-service. Each biological experiment entailed an iTRAQ 4-plex proteome comparison of immunodepleted plasma provided as 30 labeled-peptide fractions. Identical samples were analyzed by two AB SCIEX TripleTOF 5600 and three Thermo Orbitrap (Elite/Velos Pro/Q Exactive) instruments. 480 LC-MS/MS runs delivered >250 GB of data over two months. We compare herein routine service analyses of three peptide fractions of different peptide abundance. Data files from each instrument were studied to develop optimal analysis parameters to compare with default parameters in Mascot Distiller 2.4, ProteinPilot 4.5 beta, AB Sciex MS Data Converter 1.3 beta, and Proteome Discover 1.3. Peak-picking for TripleTOFs was best by ProteinPilot 4.5 beta while Mascot Distiller and Proteome Discoverer were comparable for the Orbitraps. We compared protein identification and quantitation in SwissProt 2012_07 database by Mascot Server 2.4.01 versus ProteinPilot. By all search methods, more proteins, up to two fold, were identified using the Q Exactive than others. Q Exactive excelled also at the number of unique significant peptide ion sequences. However, software-dependent impact on subsequent interpretation, due to peptide modifications, can be critical. These findings may have special implications for iTRAQ plasma proteomics. For the low abundance peptide ions, the slope of the dynamic range drop-off in the plasma proteome is uniquely sharp compared with cell lysates. Our study provides data for testable improvements in the operation of these mass spectrometers. More importantly, we have demonstrated a new affordable expedient workflow for investigators to perform proteomic experiments through the ABRF infrastructure. (We acknowledge John Cottrell for optimizing the peak-picking parameters for Mascot Distiller).

Halobacterium salinarum NRC-1 PeptideAtlas: strategies for targeted proteomics

PubMed Central

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

[Techniques for rapid production of monoclonal antibodies for use with antibody technology].

PubMed

Kamada, Haruhiko

2012-01-01

A monoclonal antibody (Mab), due to its specific binding ability to a target protein, can potentially be one of the most useful tools for the functional analysis of proteins in recent proteomics-based research. However, the production of Mab is a very time-consuming and laborious process (i.e., preparation of recombinant antigens, immunization of animals, preparation of hybridomas), making it the rate-limiting step in using Mabs in high-throughput proteomics research, which heavily relies on comprehensive and rapid methods. Therefore, there is a great demand for new methods to efficiently generate Mabs against a group of proteins identified by proteome analysis. Here, we describe a useful method called "Antibody proteomic technique" for the rapid generations of Mabs to pharmaceutical target, which were identified by proteomic analyses of disease samples (ex. tumor tissue, etc.). We also introduce another method to find profitable targets on vasculature, which is called "Vascular proteomic technique". Our results suggest that this method for the rapid generation of Mabs to proteins may be very useful in proteomics-based research as well as in clinical applications.

The NEDD8 inhibitor MLN4924 increases the size of the nucleolus and activates p53 through the ribosomal-Mdm2 pathway.

PubMed

Bailly, A; Perrin, A; Bou Malhab, L J; Pion, E; Larance, M; Nagala, M; Smith, P; O'Donohue, M-F; Gleizes, P-E; Zomerdijk, J; Lamond, A I; Xirodimas, D P

2016-01-28

The ubiquitin-like molecule NEDD8 is essential for viability, growth and development, and is a potential target for therapeutic intervention. We found that the small molecule inhibitor of NEDDylation, MLN4924, alters the morphology and increases the surface size of the nucleolus in human and germline cells of Caenorhabditis elegans in the absence of nucleolar fragmentation. SILAC proteomics and monitoring of rRNA production, processing and ribosome profiling shows that MLN4924 changes the composition of the nucleolar proteome but does not inhibit RNA Pol I transcription. Further analysis demonstrates that MLN4924 activates the p53 tumour suppressor through the RPL11/RPL5-Mdm2 pathway, with characteristics of nucleolar stress. The study identifies the nucleolus as a target of inhibitors of NEDDylation and provides a mechanism for p53 activation upon NEDD8 inhibition. It also indicates that targeting the nucleolar proteome without affecting nucleolar transcription initiates the required signalling events for the control of cell cycle regulators.

Proteomic Analysis of Excretory-Secretory Products of Mesocestoides corti Metacestodes Reveals Potential Suppressors of Dendritic Cell Functions

PubMed Central

Vendelova, Emilia; Camargo de Lima, Jeferson; Lorenzatto, Karina Rodrigues; Monteiro, Karina Mariante; Mueller, Thomas; Veepaschit, Jyotishman; Grimm, Clemens; Brehm, Klaus; Hrčková, Gabriela; Lutz, Manfred B.; Ferreira, Henrique B.

2016-01-01

Accumulating evidences have assigned a central role to parasite-derived proteins in immunomodulation. Here, we report on the proteomic identification and characterization of immunomodulatory excretory-secretory (ES) products from the metacestode larva (tetrathyridium) of the tapeworm Mesocestoides corti (syn. M. vogae). We demonstrate that ES products but not larval homogenates inhibit the stimuli-driven release of the pro-inflammatory, Th1-inducing cytokine IL-12p70 by murine bone marrow-derived dendritic cells (BMDCs). Within the ES fraction, we biochemically narrowed down the immunosuppressive activity to glycoproteins since active components were lipid-free, but sensitive to heat- and carbohydrate-treatment. Finally, using bioassay-guided chromatographic analyses assisted by comparative proteomics of active and inactive fractions of the ES products, we defined a comprehensive list of candidate proteins released by M. corti tetrathyridia as potential suppressors of DC functions. Our study provides a comprehensive library of somatic and ES products and highlight some candidate parasite factors that might drive the subversion of DC functions to facilitate the persistence of M. corti tetrathyridia in their hosts. PMID:27736880

Effect of MK-801 and Clozapine on the Proteome of Cultured Human Oligodendrocytes

PubMed Central

Cassoli, Juliana S.; Iwata, Keiko; Steiner, Johann; Guest, Paul C.; Turck, Christoph W.; Nascimento, Juliana M.; Martins-de-Souza, Daniel

2016-01-01

Separate lines of evidence have demonstrated the involvement of N-methyl-D-aspartate (NMDA) receptor and oligodendrocyte dysfunctions in schizophrenia. Here, we have carried out shotgun mass spectrometry proteome analysis of oligodendrocytes treated with the NMDA receptor antagonist MK-801 to gain potential insights into these effects at the molecular level. The MK-801 treatment led to alterations in the levels of 68 proteins, which are associated with seven distinct biological processes. Most of these proteins are involved in energy metabolism and many have been found to be dysregulated in previous proteomic studies of post-mortem brain tissues from schizophrenia patients. Finally, addition of the antipsychotic clozapine to MK-801-treated oligodendrocyte cultures resulted in changes in the levels of 45 proteins and treatment with clozapine alone altered 122 proteins and many of these showed opposite changes to the MK-801 effects. Therefore, these proteins and the associated energy metabolism pathways should be explored as potential biomarkers of antipsychotic efficacy. In conclusion, MK-801 treatment of oligodendrocytes may provide a useful model for testing the efficacy of novel treatment approaches. PMID:26973466

CPTAC Accelerates Precision Proteomics Biomedical Research | Office of Cancer Clinical Proteomics Research

Cancer.gov

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.

Proteomic changes involved in tenderization of bovine Longissimus dorsi muscle during prolonged ageing.

PubMed

Polati, Rita; Menini, Michele; Robotti, Elisa; Millioni, Renato; Marengo, Emilio; Novelli, Enrico; Balzan, Stefania; Cecconi, Daniela

2012-12-01

To study proteomic changes involved in tenderization of bovine Longissimus dorsi four Charolaise heifers and four Charolaise bull's muscles were sampled at slaughter after early and long ageing (2-4°C for 12 and 26days respectively). Descriptive sensory evaluation of samples were performed and their tenderness evaluated by Warner-Bratzler shear force test. Protein composition of fresh muscle and of meat aged was analysed by cartesian and polar 2-D electrophoresis. Student's t-test and Ranking-PCA analyses were performed to detect proteomic modulation, and the selected protein spots were identified by nano-HPLC-Chip MS/MS. This research has demonstrated that there are no differences between proteomic patterns of male and females Longissimus dorsi muscle, and that the extension of ageing beyond 12days, did not brings any concrete advantage in terms of sensory quality. Furthermore, the data presented here demonstrated that meat maturation caused changes of the abundance of proteins involved in metabolic, structural, and stress related processes. Copyright © 2012 Elsevier Ltd. All rights reserved.

Proteomics reveals novel components of the Anopheles gambiae eggshell

PubMed Central

Amenya, Dolphine A.; Chou, Wayne; Li, Jianyong; Yan, Guiyun; Gershon, Paul D.; James, Anthony A.; Marinotti, Osvaldo

2010-01-01

While genome and transcriptome sequencing has revealed a large number and diversity of Anopheles gambiae predicted proteins, identifying their functions and biosynthetic pathways remains challenging. Applied mass spectrometry based proteomics in conjunction with mosquito genome and transcriptome databases were used to identify 44 proteins as putative components of the eggshell. Among the identified molecules are two vitelline membrane proteins and a group of seven putative chorion proteins. Enzymes with peroxidase, laccase and phenoloxidase activities, likely involved in cross-linking reactions that stabilize the eggshell structure, also were identified. Seven odorant binding proteins were found in association with the mosquito eggshell, although their role has yet to be demonstrated. This analysis fills a considerable gap of knowledge about proteins that build the eggshell of anopheline mosquitoes. PMID:20433845

Michael T. Guarnieri | NREL

Science.gov Websites

accumulation," J. Proteomics (2013) "Comparative Proteomics Lends Insight into Genotype-Specific Pathogenicity," J. Proteomics (2013) "De Novo Transcriptomic Analysis of Hydrogen Production in the amino acid changes in the small envelope protein and rescued by a novel glycosolation site," J

Principles of proteome allocation are revealed using proteomic data and genome-scale models

PubMed Central

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

Proteomics in Argentina - limitations and future perspectives: A special emphasis on meat proteomics.

PubMed

Fadda, Silvina; Almeida, André M

2015-11-01

Argentina is one of the most relevant countries in Latin America, playing a major role in regional economics, culture and science. Over the last 80 years, Argentinean history has been characterized by several upward and downward phases that had major consequences on the development of science in the country and most recently on proteomics. In this article, we characterize the evolution of Proteomics sciences in Argentina over the last decade and a half. We describe the proteomics publication output of the country in the framework of the regional and international contexts, demonstrating that Argentina is solidly anchored in a regional context, showing results similar to other emergent and Latin American countries, albeit still far from the European, American or Australian realities. We also provide a case-study on the importance of Proteomics to a specific sector in the area of food science: the use of bacteria of technological interest, highlighting major achievements obtained by Argentinean proteomics scientists. Finally, we provide a general picture of the endeavors being undertaken by Argentinean Proteomics scientists and their international collaborators to promote the Proteomics-based research with the new generation of scientists and PhD students in both Argentina and other countries in the Southern cone. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Principles of proteome allocation are revealed using proteomic data and genome-scale models

DOE PAGES

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

Virtual Labs in proteomics: new E-learning tools.

PubMed

Ray, Sandipan; Koshy, Nicole Rachel; Reddy, Panga Jaipal; Srivastava, Sanjeeva

2012-05-17

Web-based educational resources have gained enormous popularity recently and are increasingly becoming a part of modern educational systems. Virtual Labs are E-learning platforms where learners can gain the experience of practical experimentation without any direct physical involvement on real bench work. They use computerized simulations, models, videos, animations and other instructional technologies to create interactive content. Proteomics being one of the most rapidly growing fields of the biological sciences is now an important part of college and university curriculums. Consequently, many E-learning programs have started incorporating the theoretical and practical aspects of different proteomic techniques as an element of their course work in the form of Video Lectures and Virtual Labs. To this end, recently we have developed a Virtual Proteomics Lab at the Indian Institute of Technology Bombay, which demonstrates different proteomics techniques, including basic and advanced gel and MS-based protein separation and identification techniques, bioinformatics tools and molecular docking methods, and their applications in different biological samples. This Tutorial will discuss the prominent Virtual Labs featuring proteomics content, including the Virtual Proteomics Lab of IIT-Bombay, and E-resources available for proteomics study that are striving to make proteomic techniques and concepts available and accessible to the student and research community. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 14). Details can be found at: http://www.proteomicstutorials.org/. Copyright © 2012 Elsevier B.V. All rights reserved.

Advances of Proteomic Sciences in Dentistry

PubMed Central

Khurshid, Zohaib; Zohaib, Sana; Najeeb, Shariq; Zafar, Muhammad Sohail; Rehman, Rabia; Rehman, Ihtesham Ur

2016-01-01

Applications of proteomics tools revolutionized various biomedical disciplines such as genetics, molecular biology, medicine, and dentistry. The aim of this review is to highlight the major milestones in proteomics in dentistry during the last fifteen years. Human oral cavity contains hard and soft tissues and various biofluids including saliva and crevicular fluid. Proteomics has brought revolution in dentistry by helping in the early diagnosis of various diseases identified by the detection of numerous biomarkers present in the oral fluids. This paper covers the role of proteomics tools for the analysis of oral tissues. In addition, dental materials proteomics and their future directions are discussed. PMID:27187379

Recent advances in proteomics of cereals.

PubMed

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.

Proteomics Analysis of Bladder Cancer Exosomes*

PubMed Central

Welton, Joanne L.; Khanna, Sanjay; Giles, Peter J.; Brennan, Paul; Brewis, Ian A.; Staffurth, John; Mason, Malcolm D.; Clayton, Aled

2010-01-01

Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function. PMID:20224111

Connecting genomic alterations to cancer biology with proteomics: the NCI Clinical Proteomic Tumor Analysis Consortium.

PubMed

Ellis, Matthew J; Gillette, Michael; Carr, Steven A; Paulovich, Amanda G; Smith, Richard D; Rodland, Karin K; Townsend, R Reid; Kinsinger, Christopher; Mesri, Mehdi; Rodriguez, Henry; Liebler, Daniel C

2013-10-01

The National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium is applying the latest generation of proteomic technologies to genomically annotated tumors from The Cancer Genome Atlas (TCGA) program, a joint initiative of the NCI and the National Human Genome Research Institute. By providing a fully integrated accounting of DNA, RNA, and protein abnormalities in individual tumors, these datasets will illuminate the complex relationship between genomic abnormalities and cancer phenotypes, thus producing biologic insights as well as a wave of novel candidate biomarkers and therapeutic targets amenable to verification using targeted mass spectrometry methods. ©2013 AACR.

Rice proteome analysis: a step toward functional analysis of the rice genome.

PubMed

Komatsu, Setsuko; Tanaka, Naoki

2005-03-01

The technique of proteome analysis using 2-DE has the power to monitor global changes that occur in the protein complement of tissues and subcellular compartments. In this review, we describe construction of the rice proteome database, the cataloging of rice proteins, and the functional characterization of some of the proteins identified. Initially, proteins extracted from various tissues and organelles were separated by 2-DE and an image analyzer was used to construct a display or reference map of the proteins. The rice proteome database currently contains 23 reference maps based on 2-DE of proteins from different rice tissues and subcellular compartments. These reference maps comprise 13 129 rice proteins, and the amino acid sequences of 5092 of these proteins are entered in the database. Major proteins involved in growth or stress responses have been identified by using a proteomics approach and some of these proteins have unique functions. Furthermore, initial work has also begun on analyzing the phosphoproteome and protein-protein interactions in rice. The information obtained from the rice proteome database will aid in the molecular cloning of rice genes and in predicting the function of unknown proteins.

Placental Proteomics: A Shortcut to Biological Insight

PubMed Central

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

Application of an Improved Proteomics Method for Abundant Protein Cleanup: Molecular and Genomic Mechanisms Study in Plant Defense*

PubMed Central

Zhang, Yixiang; Gao, Peng; Xing, Zhuo; Jin, Shumei; Chen, Zhide; Liu, Lantao; Constantino, Nasie; Wang, Xinwang; Shi, Weibing; Yuan, Joshua S.; Dai, Susie Y.

2013-01-01

High abundance proteins like ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) impose a consistent challenge for the whole proteome characterization using shot-gun proteomics. To address this challenge, we developed and evaluated Polyethyleneimine Assisted Rubisco Cleanup (PARC) as a new method by combining both abundant protein removal and fractionation. The new approach was applied to a plant insect interaction study to validate the platform and investigate mechanisms for plant defense against herbivorous insects. Our results indicated that PARC can effectively remove Rubisco, improve the protein identification, and discover almost three times more differentially regulated proteins. The significantly enhanced shot-gun proteomics performance was translated into in-depth proteomic and molecular mechanisms for plant insect interaction, where carbon re-distribution was used to play an essential role. Moreover, the transcriptomic validation also confirmed the reliability of PARC analysis. Finally, functional studies were carried out for two differentially regulated genes as revealed by PARC analysis. Insect resistance was induced by over-expressing either jacalin-like or cupin-like genes in rice. The results further highlighted that PARC can serve as an effective strategy for proteomics analysis and gene discovery. PMID:23943779

Improved chip design for integrated solid-phase microextraction in on-line proteomic sample preparation.

PubMed

Bergkvist, Jonas; Ekström, Simon; Wallman, Lars; Löfgren, Mikael; Marko-Varga, György; Nilsson, Johan; Laurell, Thomas

2002-04-01

A recently introduced silicon microextraction chip (SMEC), used for on-line proteomic sample preparation, has proved to facilitate the process of protein identification by sample clean up and enrichment of peptides. It is demonstrated that a novel grid-SMEC design improves the operating characteristics for solid-phase microextraction, by reducing dispersion effects and thereby improving the sample preparation conditions. The structures investigated in this paper are treated both numerically and experimentally. The numerical approach is based on finite element analysis of the microfluidic flow in the microchip. The analysis is accomplished by use of the computational fluid dynamics-module FLOTRAN in the ANSYS software package. The modeling and analysis of the previously reported weir-SMEC design indicates some severe drawbacks, that can be reduced by changing the microextraction chip geometry to the grid-SMEC design. The overall analytical performance was thereby improved and also verified by experimental work. Matrix-assisted laser desorption/ionization mass spectra of model peptides extracted from both the weir-SMEC and the new grid-SMEC support the numerical analysis results. Further use of numerical modeling and analysis of the SMEC structures is also discussed and suggested in this work.

Gingival crevicular fluid proteomes in health, gingivitis and chronic periodontitis.

PubMed

Huynh, A H S; Veith, P D; McGregor, N R; Adams, G G; Chen, D; Reynolds, E C; Ngo, L H; Darby, I B

2015-10-01

The aim of this study was to compare the proteome composition of gingival crevicular fluid obtained from healthy periodontium, gingivitis and chronic periodontitis affected sites. Owing to its site-specific nature, gingival crevicular fluid is ideal for studying biological processes that occur during periodontal health and disease progression. However, few studies have been conducted into the gingival crevicular fluid proteome due to the small volumes obtained. Fifteen males were chosen for each of three different groups, healthy periodontium, gingivitis and chronic periodontitis. They were categorized based on clinical measurements including probing depth, bleeding on probing, plaque index, radiographic bone level, modified gingival index and smoking status. Gingival crevicular fluid was collected from each patient, pooled into healthy, gingivitis and chronic periodontitis groups and their proteome analyzed by gel electrophoresis and liquid chromatography electrospray ionization ion trap tandem mass spectrometry. One hundred and twenty-one proteins in total were identified, and two-thirds of these were identified in all three conditions. Forty-two proteins were considered to have changed in abundance. Of note, cystatin B and cystatin S decreased in abundance from health to gingivitis and further in chronic periodontitis. Complement proteins demonstrated an increase from health to gingivitis followed by a decrease in chronic periodontitis. Immunoglobulins, keratin proteins, fibronectin, lactotransferrin precursor, 14-3-3 protein zeta/delta, neutrophil defensin 3 and alpha-actinin exhibited fluctuations in levels. The gingival crevicular fluid proteome in each clinical condition was different and its analysis may assist us in understanding periodontal pathogenesis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Search for novel circulating cancer chemopreventive biomarkers of dietary rice bran intervention in Apc(Min) mice model of colorectal carcinogenesis, using proteomic and metabolic profiling strategies.

PubMed

Norris, Leonie; Malkar, Aditya; Horner-Glister, Emma; Hakimi, Amirmansoor; Ng, Leong L; Gescher, Andreas J; Creaser, Colin; Sale, Stewart; Jones, Donald J L

2015-09-01

There is strong epidemiological evidence indicating that consumption by humans of whole-grain foods including rice bran may be associated with a low incidence of cancer, especially in the colorectum. Molecular processes associated with cancer development may be retarded by fiber consumption. Consequently, intervention with dietary fiber might be suitable as a cancer chemoprevention strategy in high-risk populations. Here, we searched for putative molecular mechanism-based efficacy biomarkers of rice fiber consumption in the plasma of mice characterized by a genetic propensity to develop gastrointestinal adenomas. The hypothesis was tested that metabolic and proteomic changes in blood reflect the chemopreventive activity of rice bran. Apc(Min) mice received diet supplemented with rice bran at 5, 15, and 30%. Blood and tissue samples were taken. Plasma was subjected to MS-based proteomic and metabolic profiling analyses as well as assessment of hematocrit values. Gastrointestinal tracts were removed and adenomas were counted and their size was measured so that total tumor burden could be calculated. The hypothesis was tested that metabolic and proteomic changes in blood reflect chemopreventive activity. Rice bran consumption reduced adenoma burden and number in a dose-related fashion when compared to controls. Metabolic profiling data demonstrated strong clustering of the groups indicating that metabolic pathways are perturbed. Proteomic analysis identified adiponectin as a molecule that was significantly altered, which may play a role in tumor suppression. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differential Denaturation of Serum Proteome Reveals a Significant Amount of Hidden Information in Complex Mixtures of Proteins

PubMed Central

Polci, Maria Letizia; Rossi, Stefania; Cordella, Martina; Carlucci, Giuseppe; Marchetti, Paolo; Antonini-Cappellini, Giancarlo; Facchiano, Antonio; D'Arcangelo, Daniela; Facchiano, Francesco

2013-01-01

Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different denaturation treatments were tested and the 3 most discriminating ones were selected (TRIDENT analysis) and applied to human sera, showing a significant improvement of serum protein discrimination as confirmed by MALDI-TOF/MS and LC-MS/MS identification, depending on the type of denaturation applied. Thereafter sera from mice and patients carrying cutaneous melanoma were analyzed through TRIDENT. Nine and 8 protein bands were found differentially expressed in mice and human melanoma sera, compared to healthy controls (p<0.05); three of them were found, for the first time, significantly modulated: α2macroglobulin (down-regulated in melanoma, p<0.001), Apolipoprotein-E and Apolipoprotein-A1 (both up-regulated in melanoma, p<0.04), both in mice and humans. The modulation was confirmed by immunological methods. Other less abundant proteins (e.g. gelsolin) were found significantly modulated (p<0.05). Conclusions: i) serum proteome contains a large amount of information, still neglected, related to proteins folding; ii) a careful serum denaturation may significantly improve analytical procedures involving complex protein mixtures; iii) serum differential denaturation protocol highlights interesting proteomic differences between cancer and healthy sera. PMID:23533572

Analysis of essential gene dynamics under antibiotic stress in Streptococcus sanguinis

PubMed Central

El-Rami, Fadi; Kong, Xiangzhen; Parikh, Hardik; Zhu, Bin; Stone, Victoria; Kitten, Todd; Xu, Ping

2018-01-01

The paradoxical response of Streptococcus sanguinis to drugs prescribed for dental and clinical practices has complicated treatment guidelines and raised the need for further investigation. We conducted a high throughput study on concomitant transcriptome and proteome dynamics in a time course to assess S. sanguinis behaviour under a sub-inhibitory concentration of ampicillin. Temporal changes at the transcriptome and proteome level were monitored to cover essential genes and proteins over a physiological map of intricate pathways. Our findings revealed that translation was the functional category in S. sanguinis that was most enriched in essential proteins. Moreover, essential proteins in this category demonstrated the greatest conservation across 2774 bacterial proteomes, in comparison to other essential functional categories like cell wall biosynthesis and energy production. In comparison to non-essential proteins, essential proteins were less likely to contain ‘degradation-prone’ amino acids at their N-terminal position, suggesting a longer half-life. Despite the ampicillin-induced stress, the transcriptional up-regulation of amino acid-tRNA synthetases and proteomic elevation of amino acid biosynthesis enzymes favoured the enriched components of essential proteins revealing ‘proteomic signatures’ that can be used to bridge the genotype–phenotype gap of S. sanguinis under ampicillin stress. Furthermore, we identified a significant correlation between the levels of mRNA and protein for essential genes and detected essential protein-enriched pathways differentially regulated through a persistent stress response pattern at late time points. We propose that the current findings will help characterize a bacterial model to study the dynamics of essential genes and proteins under clinically relevant stress conditions. PMID:29393020

Excess copper induces anoxygenic photosynthesis in Anabaena doliolum: a homology based proteomic assessment of its survival strategy.

PubMed

Bhargava, Poonam; Mishra, Yogesh; Srivastava, Ashish Kumar; Narayan, Om Prakash; Rai, Lal Chand

2008-04-01

This study is the first to demonstrate operation of anoxygenic photosynthesis in copper acclimated Anabaena doliolum and to offer proteomic comparison with the control cells. The Cu-treated control strain showed a negative correlation in growth and intracellular Cu, partial inhibition of O(2)-evolution, PS II, PS I, whole chain, chlorophyll absorption, and nitrogenase activity. However, the acclimated strain growing in 250-fold excess Cu exhibited near normal growth, ATP content, PS I activity, carbon fixation, and almost complete inhibition of O(2)-evolution, PS II and chlorophyll absorption, but increased nitrogenase activity as compared to control. Proteomic decoding of the survival strategy of Cu-treated control and the acclimated strain using two-dimensional gel electrophoresis and MALDI-TOF MS analysis of proteins displaying significant and reproducible changes demonstrated involvement of transketolase, phycoerythrocyanin alpha-chain, iron superoxide dismutase (Fe-SOD), hypothetical protein alr 0803, manganese superoxide dismutase (Mn-SOD), phosphoribulokinase, and plastocyanin (PLC). Expression pattern of these proteins was attested at the transcriptional level using RT-PCR. Time course analysis of proteins of Cu-treated control strain revealed almost no change in PLC level, and a minor accumulation of transketolase, phycoerythrocyanin alpha-chain and both isoforms of SOD after 7 and recovery after 10 days. Acclimated strain under excess Cu, however, exhibited significant accumulation of both isoforms of SOD, plastocyanin, phosphoribulokinase and transketolase, which seem to counteract oxidative damage, serve as an alternate electron carrier from cytochrome b6/f complex to photosystem I and meet the NADPH and ATP requirements, respectively, under anoxygenic photosynthesis. In view of the kinetics of the hypothetical protein alr0803 (no change in expression level for 7, maximum after 10 and decline after 15 days) its involvement in metal homeostasis is suggested.

Extensive proteomic remodeling is induced by eukaryotic translation elongation factor 1Bγ deletion in Aspergillus fumigatus.

PubMed

O'Keeffe, Grainne; Jöchl, Christoph; Kavanagh, Kevin; Doyle, Sean

2013-11-01

The opportunistic pathogen Aspergillus fumigatus is ubiquitous in the environment and predominantly infects immunocompromised patients. The functions of many genes remain unknown despite sequencing of the fungal genome. A putative translation elongation factor 1Bγ (eEF1Bγ, termed elfA; 750 bp) is expressed, and exhibits glutathione S-transferase activity, in A. fumigatus. Here, we demonstrate the role of ElfA in the oxidative stress response, as well as a possible involvement in translation and actin cytoskeleton organization, respectively. Comparative proteomics, in addition to phenotypic analysis, under basal and oxidative stress conditions, demonstrated a role for A. fumigatus elfA in the oxidative stress response. An elfA-deficient strain (A. fumigatus ΔelfA) was significantly more sensitive to the oxidants H2O2, diamide, and 4,4'-dipyridyl disulfide (DPS) than the wild-type. This was further supported with the identification of differentially expressed proteins of the oxidative stress response, including; mitochondrial peroxiredoxin Prx1, molecular chaperone Hsp70 and mitochondrial glycerol-3-phosphate dehydrogenase. Phenotypic analysis also revealed that A. fumigatus ΔelfA was significantly more tolerant to voriconazole than the wild-type. The differential expression of two aminoacyl-tRNA synthetases suggests a role for A. fumigatus elfA in translation, while the identification of actin-bundling protein Sac6 and vacuolar dynamin-like GTPase VpsA link A. fumigatus elfA to the actin cytoskeleton. Overall, this work highlights the diverse roles of A. fumigatus elfA, with respect to translation, oxidative stress and actin cytoskeleton organization. In addition to this, the strategy of combining targeted gene deletion with comparative proteomics for elucidating the role of proteins of unknown function is further revealed. © 2013 The Protein Society.

Quantitative proteomics in teleost fish: insights and challenges for neuroendocrine and neurotoxicology research.

PubMed

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 regulation of neuronal stability and structure. Lastly, for fish neuroendocrine studies using both high-throughput genomics and proteomics, we compare gene and protein relationships in the hypothalamus and demonstrate that correlation is often poor for single time point experiments. These studies highlight the need for additional time course analyses to better understand gene-protein relationships and adverse outcome pathways. This is important if both transcriptomics and proteomics are to be used together to investigate neuroendocrine signaling pathways or as bio-monitoring tools in ecotoxicology. Copyright © 2011 Elsevier Inc. All rights reserved.

Development of Droplet Microfluidics Enabling High-Throughput Single-Cell Analysis.

PubMed

Wen, Na; Zhao, Zhan; Fan, Beiyuan; Chen, Deyong; Men, Dong; Wang, Junbo; Chen, Jian

2016-07-05

This article reviews recent developments in droplet microfluidics enabling high-throughput single-cell analysis. Five key aspects in this field are included in this review: (1) prototype demonstration of single-cell encapsulation in microfluidic droplets; (2) technical improvements of single-cell encapsulation in microfluidic droplets; (3) microfluidic droplets enabling single-cell proteomic analysis; (4) microfluidic droplets enabling single-cell genomic analysis; and (5) integrated microfluidic droplet systems enabling single-cell screening. We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on key performances of throughput, multifunctionality, and absolute quantification.

Recent advances on multidimensional liquid chromatography-mass spectrometry for proteomics: from qualitative to quantitative analysis--a review.

PubMed

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.

Proteomic analysis of tissue samples in translational breast cancer research.

PubMed

Gromov, Pavel; Moreira, José M A; Gromova, Irina

2014-06-01

In the last decade, many proteomic technologies have been applied, with varying success, to the study of tissue samples of breast carcinoma for protein expression profiling in order to discover protein biomarkers/signatures suitable for: characterization and subtyping of tumors; early diagnosis, and both prognosis and prediction of outcome of chemotherapy. The purpose of this review is to critically appraise what has been achieved to date using proteomic technologies and to bring forward novel strategies - based on the analysis of clinically relevant samples - that promise to accelerate the translation of basic discoveries into the daily breast cancer clinical practice. In particular, we address major issues in experimental design by reviewing the strengths and weaknesses of current proteomic strategies in the context of the analysis of human breast tissue specimens.

From proteomics to systems biology: MAPA, MASS WESTERN, PROMEX, and COVAIN as a user-oriented platform.

PubMed

Weckwerth, Wolfram; Wienkoop, Stefanie; Hoehenwarter, Wolfgang; Egelhofer, Volker; Sun, Xiaoliang

2014-01-01

Genome sequencing and systems biology are revolutionizing life sciences. Proteomics emerged as a fundamental technique of this novel research area as it is the basis for gene function analysis and modeling of dynamic protein networks. Here a complete proteomics platform suited for functional genomics and systems biology is presented. The strategy includes MAPA (mass accuracy precursor alignment; http://www.univie.ac.at/mosys/software.html ) as a rapid exploratory analysis step; MASS WESTERN for targeted proteomics; COVAIN ( http://www.univie.ac.at/mosys/software.html ) for multivariate statistical analysis, data integration, and data mining; and PROMEX ( http://www.univie.ac.at/mosys/databases.html ) as a database module for proteogenomics and proteotypic peptides for targeted analysis. Moreover, the presented platform can also be utilized to integrate metabolomics and transcriptomics data for the analysis of metabolite-protein-transcript correlations and time course analysis using COVAIN. Examples for the integration of MAPA and MASS WESTERN data, proteogenomic and metabolic modeling approaches for functional genomics, phosphoproteomics by integration of MOAC (metal-oxide affinity chromatography) with MAPA, and the integration of metabolomics, transcriptomics, proteomics, and physiological data using this platform are presented. All software and step-by-step tutorials for data processing and data mining can be downloaded from http://www.univie.ac.at/mosys/software.html.

N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana *

PubMed Central

Ndah, Elvis; Jonckheere, Veronique

2017-01-01

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes. PMID:28432195

N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana.

PubMed

Willems, Patrick; Ndah, Elvis; Jonckheere, Veronique; Stael, Simon; Sticker, Adriaan; Martens, Lennart; Van Breusegem, Frank; Gevaert, Kris; Van Damme, Petra

2017-06-01

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteome analysis of the Mycobacterium tuberculosis Beijing B0/W148 cluster

PubMed Central

Bespyatykh, Julia; Shitikov, Egor; Butenko, Ivan; Altukhov, Ilya; Alexeev, Dmitry; Mokrousov, Igor; Dogonadze, Marine; Zhuravlev, Viacheslav; Yablonsky, Peter; Ilina, Elena; Govorun, Vadim

2016-01-01

Beijing B0/W148, a “successful” clone of Mycobacterium tuberculosis, is widespread in the Russian Federation and some countries of the former Soviet Union. Here, we used label-free gel-LC-MS/MS shotgun proteomics to discover features of Beijing B0/W148 strains that could explain their success. Qualitative and quantitative proteome analyses of Beijing B0/W148 strains allowed us to identify 1,868 proteins, including 266 that were differentially abundant compared with the control strain H37Rv. To predict the biological effects of the observed differences in protein abundances, we performed Gene Ontology analysis together with analysis of protein-DNA interactions using a gene regulatory network. Our results demonstrate that Beijing B0/W148 strains have increased levels of enzymes responsible for long-chain fatty acid biosynthesis, along with a coincident decrease in the abundance of proteins responsible for their degradation. Together with high levels of HsaA (Rv3570c) protein, involved in steroid degradation, these findings provide a possible explanation for the increased transmissibility of Beijing B0/W148 strains and their survival in host macrophages. Among other, we confirmed a very low level of the SseA (Rv3283) protein in Beijing B0/W148 characteristic for all «modern» Beijing strains, which could lead to increased DNA oxidative damage, accumulation of mutations, and potentially facilitate the development of drug resistance. PMID:27356881

SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries.

PubMed

Wu, Jemma X; Song, Xiaomin; Pascovici, Dana; Zaw, Thiri; Care, Natasha; Krisp, Christoph; Molloy, Mark P

2016-07-01

The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries*

PubMed Central

Wu, Jemma X.; Song, Xiaomin; Pascovici, Dana; Zaw, Thiri; Care, Natasha; Krisp, Christoph; Molloy, Mark P.

2016-01-01

The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries. PMID:27161445

Highly efficient proteome analysis with combination of protein pre-fractionation by preparative microscale solution isoelectric focusing and identification by μRPLC-MS/MS with serially coupled long microcolumn.

PubMed

Tao, Dingyin; Sun, Liangliang; Zhu, Guijie; Liang, Yu; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

2011-01-01

To improve the efficiency of proteome analysis, a strategy with the combination of protein pre-fractionation by preparative microscale solution isoelectric focusing, peptide separation by μRPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS was proposed. By preparative microscale solution isoelectric focusing technique, proteins extracted from whole cell lysates of Escherichia coli were fractionated into five chambers divided by isoelectric membranes, respectively with pH range from 3.0 to 4.6, 4.6 to 5.4, 5.4 to 6.2, 6.2 to 7.0 and 7.0 to 10.0. Compared to the traditional on-gel IFF, the protein recovery could be obviously improved to over 95%. Subsequently, the enriched and fractionated proteins in each chamber were digested, and further separated by a 30-cm long serially coupled RP microcolumn. Through the detection by ESI-MS/MS, about 200 proteins were identified in each fraction, and in total 835 proteins were identified even with one-dimensional μRPLC-MS/MS system. All these results demonstrate that by such a combination strategy, highly efficient proteome analysis could be achieved, not only due to the in-solution protein enrichment and pre-fractionation with improved protein recovery but also owing to the increased separation capacity of serially coupled long μRPLC columns. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enrichment and proteomic analysis of plasma membrane from rat dorsal root ganglions

PubMed Central

2009-01-01

Background Dorsal root ganglion (DRG) neurons are primary sensory neurons that conduct neuronal impulses related to pain, touch and temperature senses. Plasma membrane (PM) of DRG cells plays important roles in their functions. PM proteins are main performers of the functions. However, mainly due to the very low amount of DRG that leads to the difficulties in PM sample collection, few proteomic analyses on the PM have been reported and it is a subject that demands further investigation. Results By using aqueous polymer two-phase partition in combination with high salt and high pH washing, PMs were efficiently enriched, demonstrated by western blot analysis. A total of 954 non-redundant proteins were identified from the plasma membrane-enriched preparation with CapLC-MS/MS analysis subsequent to protein separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or shotgun digestion. 205 (21.5%) of the identified proteins were unambiguously assigned as PM proteins, including a large number of signal proteins, receptors, ion channel and transporters. Conclusion The aqueous polymer two-phase partition is a simple, rapid and relatively inexpensive method. It is well suitable for the purification of PMs from small amount of tissues. Therefore, it is reasonable for the DRG PM to be enriched by using aqueous two-phase partition as a preferred method. Proteomic analysis showed that DRG PM was rich in proteins involved in the fundamental biological processes including material exchange, energy transformation and information transmission, etc. These data would help to our further understanding of the fundamental DRG functions. PMID:19889238

Identification and proteomic analysis of osteoblast-derived exosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ge, Min; Ke, Ronghu; Cai, Tianyi

Exosomes are nanometer-sized vesicles with the function of intercellular communication, and they are released by various cell types. To reveal the knowledge about the exosomes from osteoblast, and explore the potential functions of osteogenesis, we isolated microvesicles from supernatants of mouse Mc3t3 by ultracentrifugation, characterized exosomes by electron microscopy and immunoblotting and presented the protein profile by proteomic analysis. The result demonstrated that microvesicles were between 30 and 100 nm in diameter, round shape with cup-like concavity and expressed exosomal marker tumor susceptibility gene (TSG) 101 and flotillin (Flot) 1. We identified a total number of 1069 proteins among which 786more » proteins overlap with ExoCarta database. Gene Oncology analysis indicated that exosomes mostly derived from plasma membrane and mainly involved in protein localization and intracellular signaling. The Ingenuity Pathway Analysis showed pathways are mostly involved in exosome biogenesis, formation, uptake and osteogenesis. Among the pathways, eukaryotic initiation factor 2 pathways played an important role in osteogenesis. Our study identified osteoblast-derived exosomes, unveiled the content of them, presented potential osteogenesis-related proteins and pathways and provided a rich proteomics data resource that will be valuable for further studies of the functions of individual proteins in bone diseases. - Highlights: • We for the first time identified exosomes from mouse osteoblast. • Osteoblasts-derived exosomes contain osteoblast peculiar proteins. • Proteins from osteoblasts-derived exosomes are intently involved in EIF2 pathway. • EIF2α from the EIF2 pathway plays an important role in osteogenesis.« less

Molecular phenotype of zebrafish ovarian follicle by serial analysis of gene expression and proteomic profiling, and comparison with the transcriptomes of other animals

PubMed Central

Knoll-Gellida, Anja; André, Michèle; Gattegno, Tamar; Forgue, Jean; Admon, Arie; Babin, Patrick J

2006-01-01

Background The ability of an oocyte to develop into a viable embryo depends on the accumulation of specific maternal information and molecules, such as RNAs and proteins. A serial analysis of gene expression (SAGE) was carried out in parallel with proteomic analysis on fully-grown ovarian follicles from zebrafish (Danio rerio). The data obtained were compared with ovary/follicle/egg molecular phenotypes of other animals, published or available in public sequence databases. Results Sequencing of 27,486 SAGE tags identified 11,399 different ones, including 3,329 tags with an occurrence superior to one. Fifty-eight genes were expressed at over 0.15% of the total population and represented 17.34% of the mRNA population identified. The three most expressed transcripts were a rhamnose-binding lectin, beta-actin 2, and a transcribed locus similar to the H2B histone family. Comparison with the large-scale expressed sequence tags sequencing approach revealed highly expressed transcripts that were not previously known to be expressed at high levels in fish ovaries, like the short-sized polarized metallothionein 2 transcript. A higher sensitivity for the detection of transcripts with a characterized maternal genetic contribution was also demonstrated compared to large-scale sequencing of cDNA libraries. Ferritin heavy polypeptide 1, heat shock protein 90-beta, lactate dehydrogenase B4, beta-actin isoforms, tubulin beta 2, ATP synthase subunit 9, together with 40 S ribosomal protein S27a, were common highly-expressed transcripts of vertebrate ovary/unfertilized egg. Comparison of transcriptome and proteome data revealed that transcript levels provide little predictive value with respect to the extent of protein abundance. All the proteins identified by proteomic analysis of fully-grown zebrafish follicles had at least one transcript counterpart, with two exceptions: eosinophil chemotactic cytokine and nothepsin. Conclusion This study provides a complete sequence data set of maternal mRNA stored in zebrafish germ cells at the end of oogenesis. This catalogue contains highly-expressed transcripts that are part of a vertebrate ovarian expressed gene signature. Comparison of transcriptome and proteome data identified downregulated transcripts or proteins potentially incorporated in the oocyte by endocytosis. The molecular phenotype described provides groundwork for future experimental approaches aimed at identifying functionally important stored maternal transcripts and proteins involved in oogenesis and early stages of embryo development. PMID:16526958

Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.

PubMed

Rusin, Scott F; Schlosser, Kate A; Adamo, Mark E; Kettenbach, Arminja N

2015-10-13

Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c-dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2-dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. Copyright © 2015, American Association for the Advancement of Science.

Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells

PubMed Central

Rusin, Scott F.; Schlosser, Kate A.; Adamo, Mark E.; Kettenbach, Arminja N.

2017-01-01

Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry–based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c–dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2–dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. PMID:26462736

Proteomic analysis of mare follicular fluid during late follicle development

PubMed Central

2011-01-01

Background Follicular fluid accumulates into the antrum of follicle from the early stage of follicle development. Studies on its components may contribute to a better understanding of the mechanisms underlying follicular development and oocyte quality. With this objective, we performed a proteomic analysis of mare follicular fluid. First, we hypothesized that proteins in follicular fluid may differ from those in the serum, and also may change during follicle development. Second, we used four different approaches of Immunodepletion and one enrichment method, in order to overcome the masking effect of high-abundance proteins present in the follicular fluid, and to identify those present in lower abundance. Finally, we compared our results with previous studies performed in mono-ovulant (human) and poly-ovulant (porcine and canine) species in an attempt to identify common and/or species-specific proteins. Methods Follicular fluid samples were collected from ovaries at three different stages of follicle development (early dominant, late dominant and preovulatory). Blood samples were also collected at each time. The proteomic analysis was carried out on crude, depleted and enriched follicular fluid by 2D-PAGE, 1D-PAGE and mass spectrometry. Results Total of 459 protein spots were visualized by 2D-PAGE of crude mare follicular fluid, with no difference among the three physiological stages. Thirty proteins were observed as differentially expressed between serum and follicular fluid. Enrichment method was found to be the most powerful method for detection and identification of low-abundance proteins from follicular fluid. Actually, we were able to identify 18 proteins in the crude follicular fluid, and as many as 113 in the enriched follicular fluid. Inhibins and a few other proteins involved in reproduction could only be identified after enrichment of follicular fluid, demonstrating the power of the method used. The comparison of proteins found in mare follicular fluid with proteins previously identified in human, porcine and canine follicular fluids, led to the identification of 12 common proteins and of several species-specific proteins. Conclusions This study provides the first description of mare follicular fluid proteome during the late follicle development stages. We identified several proteins from crude, depleted and enriched follicular fluid. Our results demonstrate that the enrichment method, combined with 2D-PAGE and mass spectrometry, can be successfully used to visualize and further identify the low-abundance proteins in the follicular fluid. PMID:21923925

Proteomic analysis of mare follicular fluid during late follicle development.

PubMed

Fahiminiya, Somayyeh; Labas, Valérie; Roche, Stéphane; Dacheux, Jean-Louis; Gérard, Nadine

2011-09-17

Follicular fluid accumulates into the antrum of follicle from the early stage of follicle development. Studies on its components may contribute to a better understanding of the mechanisms underlying follicular development and oocyte quality. With this objective, we performed a proteomic analysis of mare follicular fluid. First, we hypothesized that proteins in follicular fluid may differ from those in the serum, and also may change during follicle development. Second, we used four different approaches of Immunodepletion and one enrichment method, in order to overcome the masking effect of high-abundance proteins present in the follicular fluid, and to identify those present in lower abundance. Finally, we compared our results with previous studies performed in mono-ovulant (human) and poly-ovulant (porcine and canine) species in an attempt to identify common and/or species-specific proteins. Follicular fluid samples were collected from ovaries at three different stages of follicle development (early dominant, late dominant and preovulatory). Blood samples were also collected at each time. The proteomic analysis was carried out on crude, depleted and enriched follicular fluid by 2D-PAGE, 1D-PAGE and mass spectrometry. Total of 459 protein spots were visualized by 2D-PAGE of crude mare follicular fluid, with no difference among the three physiological stages. Thirty proteins were observed as differentially expressed between serum and follicular fluid. Enrichment method was found to be the most powerful method for detection and identification of low-abundance proteins from follicular fluid. Actually, we were able to identify 18 proteins in the crude follicular fluid, and as many as 113 in the enriched follicular fluid. Inhibins and a few other proteins involved in reproduction could only be identified after enrichment of follicular fluid, demonstrating the power of the method used. The comparison of proteins found in mare follicular fluid with proteins previously identified in human, porcine and canine follicular fluids, led to the identification of 12 common proteins and of several species-specific proteins. This study provides the first description of mare follicular fluid proteome during the late follicle development stages. We identified several proteins from crude, depleted and enriched follicular fluid. Our results demonstrate that the enrichment method, combined with 2D-PAGE and mass spectrometry, can be successfully used to visualize and further identify the low-abundance proteins in the follicular fluid.

Proteomic analysis of the impacts of powdery mildew on wheat grain.

PubMed

Li, Jie; Liu, Xinhao; Yang, Xiwen; Li, Yongchun; Wang, Chenyang; He, Dexian

2018-09-30

Powdery mildew of wheat is one of the major foliar diseases, causing significant yield loss and flour quality change. In this study, grain protein and starch response to powdery mildew infection were investigated. Total protein, glutenin and gliadin exhibited a greater increase in grains from infected wheat, while the content of total starch and amylopectin was decreased. Comparative proteomic analysis demonstrated that the overabundant protein synthesis-related proteins might facilitate the accumulation of storage proteins in grains from infected plants. The significant increase in triticin, serpin and HMW-GS in grains from infected wheat might relate to the superior gluten quality. In addition, overabundant carbohydrate metabolism-related proteins in grains from infected wheat were conducive to the depletion of starch, whereas the decreased abundance of ADP glucose pyrophosphorylase might be related to the deficiency of starch synthesis. These results provide a deeper understanding on the change of wheat quality under powdery mildew infection. Copyright © 2018 Elsevier Ltd. All rights reserved.

Gene Expression and Proteome Analysis as Sources of Biomarkers in Basal Cell Carcinoma

PubMed Central

Ghita, Mihaela Adriana; Voiculescu, Suzana; Rosca, Adrian E.; Moraru, Liliana; Greabu, Maria

2016-01-01

Basal cell carcinoma (BCC) is the world's leading skin cancer in terms of frequency at the moment and its incidence continues to rise each year, leading to profound negative psychosocial and economic consequences. UV exposure is the most important environmental factor in the development of BCC in genetically predisposed individuals, this being reflected by the anatomical distribution of lesions mainly on sun-exposed skin areas. Early diagnosis and prompt management are of crucial importance in order to prevent local tissue destruction and subsequent disfigurement. Although various noninvasive or minimal invasive techniques have demonstrated their utility in increasing diagnostic accuracy of BCC and progress has been made in its treatment options, recurrent, aggressive, and metastatic variants of BCC still pose significant challenge for the healthcare system. Analysis of gene expression and proteomic profiling of tumor cells and of tumoral microenvironment in various tissues strongly suggests that certain molecules involved in skin cancer pathogenic pathways might represent novel predictive and prognostic biomarkers in BCC. PMID:27578920

Proteomic characterization of a mouse model of familial Danish dementia.

PubMed

Vitale, Monica; Renzone, Giovanni; Matsuda, Shuji; Scaloni, Andrea; D'Adamio, Luciano; Zambrano, Nicola

2012-01-01

A dominant mutation in the ITM2B/BRI2 gene causes familial Danish dementia (FDD) in humans. To model FDD in animal systems, a knock-in approach was recently implemented in mice expressing a wild-type and mutant allele, which bears the FDD-associated mutation. Since these FDD(KI) mice show behavioural alterations and impaired synaptic function, we characterized their synaptosomal proteome via two-dimensional differential in-gel electrophoresis. After identification by nanoliquid chromatography coupled to electrospray-linear ion trap tandem mass spectrometry, the differentially expressed proteins were classified according to their gene ontology descriptions and their predicted functional interactions. The Dlg4/Psd95 scaffold protein and additional signalling proteins, including protein phosphatases, were revealed by STRING analysis as potential players in the altered synaptic function of FDD(KI) mice. Immunoblotting analysis finally demonstrated the actual downregulation of the synaptosomal scaffold protein Dlg4/Psd95 and of the dual-specificity phosphatase Dusp3 in the synaptosomes of FDD(KI) mice.

Proteomic Characterization of a Mouse Model of Familial Danish Dementia

PubMed Central

Vitale, Monica; Renzone, Giovanni; Matsuda, Shuji; Scaloni, Andrea; D'Adamio, Luciano; Zambrano, Nicola

2012-01-01

A dominant mutation in the ITM2B/BRI2 gene causes familial Danish dementia (FDD) in humans. To model FDD in animal systems, a knock-in approach was recently implemented in mice expressing a wild-type and mutant allele, which bears the FDD-associated mutation. Since these FDDKI mice show behavioural alterations and impaired synaptic function, we characterized their synaptosomal proteome via two-dimensional differential in-gel electrophoresis. After identification by nanoliquid chromatography coupled to electrospray-linear ion trap tandem mass spectrometry, the differentially expressed proteins were classified according to their gene ontology descriptions and their predicted functional interactions. The Dlg4/Psd95 scaffold protein and additional signalling proteins, including protein phosphatases, were revealed by STRING analysis as potential players in the altered synaptic function of FDDKI mice. Immunoblotting analysis finally demonstrated the actual downregulation of the synaptosomal scaffold protein Dlg4/Psd95 and of the dual-specificity phosphatase Dusp3 in the synaptosomes of FDDKI mice. PMID:22619496

Proteomic analysis of high yield rice variety mutated from spaceflight

NASA Astrophysics Data System (ADS)

Ma, Y.; Cheng, Z.; Wang, W.; Sun, Y.

Seeds of pure rice varieties were flown on Chinese recoverable satellite, JB-1, for a 15-day flight in 1996. Many mutant rice varieties with various phenotypes were generated after continuous selection and breeding. Among the mutants, a variety 971-5 showed a significant increase in grain yield compared to its control (971ck). In this study, proteomic analysis of both mutant variety 971-5 and control variety 971ck were carried out to investigate the changes of protein expression level in their leaves at three different growth stages (early and middle stage of tillering, and booting stage). Results showed that (1) almost all differentially expressed proteins were down-regulated in 971-5 with only one exception, (2) the percentages of differentially expressed proteins were 3.1%, 2.1% and 3.1% at the three stages, respectively, and (3) one protein showed a significant alteration in its molecular weight (MW). These data demonstrated that the space environment can alter the expression level of rice proteins both quantitatively and qualitatively.

Proteomic analysis of the kidney filtration barrier--Problems and perspectives.

PubMed

Rinschen, Markus M; Benzing, Thomas; Limbutara, Kavee; Pisitkun, Trairak

2015-12-01

Diseases of the glomerular filter of the kidney are a leading cause of end-stage renal failure. The kidney filter is localized within the renal glomeruli, small microvascular units that are responsible for ultrafiltration of about 180 liters of primary urine every day. The renal filter consists of three layers, fenestrated endothelial cells, glomerular basement membrane, and the podocytes, terminally differentiated, arborized epithelial cells. This review demonstrates the use of proteomics to generate insights into the regulation of the renal filtration barrier at a molecular level. The advantages and disadvantages of different glomerular purification methods are examined, and the technical limitations that have been significantly improved by in silico or biochemical approaches are presented. We also comment on phosphoproteomic studies that have generated considerable molecular-level understanding of the physiological regulation of the kidney filter. Lastly, we conclude with an analysis of urinary exosomes as a potential filter-derived resource for the noninvasive discovery of glomerular disease mechanisms. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Advancing Cell Biology Through Proteomics in Space and Time (PROSPECTS)*

PubMed Central

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

Comparative analysis of inflamed and non-inflamed colon biopsies reveals strong proteomic inflammation profile in patients with ulcerative colitis

PubMed Central

2012-01-01

Background Accurate diagnostic and monitoring tools for ulcerative colitis (UC) are missing. Our aim was to describe the proteomic profile of UC and search for markers associated with disease exacerbation. Therefore, we aimed to characterize specific proteins associated with inflamed colon mucosa from patients with acute UC using mass spectrometry-based proteomic analysis. Methods Biopsies were sampled from rectum, sigmoid colon and left colonic flexure from twenty patients with active proctosigmoiditis and from four healthy controls for proteomics and histology. Proteomic profiles of whole colonic biopsies were characterized using 2D-gel electrophoresis, and peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for identification of differently expressed protein spots. Results A total of 597 spots were annotated by image analysis and 222 of these had a statistically different protein level between inflamed and non-inflamed tissue in the patient group. Principal component analysis clearly grouped non-inflamed samples separately from the inflamed samples indicating that the proteomic signature of colon mucosa with acute UC is strong. Totally, 43 individual protein spots were identified, including proteins involved in energy metabolism (triosephosphate isomerase, glycerol-3-phosphate-dehydrogenase, alpha enolase and L-lactate dehydrogenase B-chain) and in oxidative stress (superoxide dismutase, thioredoxins and selenium binding protein). Conclusions A distinct proteomic profile of inflamed tissue in UC patients was found. Specific proteins involved in energy metabolism and oxidative stress were identified as potential candidate markers for UC. PMID:22726388

Histoplasma capsulatum proteome response to decreased iron availability

PubMed Central

Winters, Michael S; Spellman, Daniel S; Chan, Qilin; Gomez, Francisco J; Hernandez, Margarita; Catron, Brittany; Smulian, Alan G; Neubert, Thomas A; Deepe, George S

2008-01-01

Background A fundamental pathogenic feature of the fungus Histoplasma capsulatum is its ability to evade innate and adaptive immune defenses. Once ingested by macrophages the organism is faced with several hostile environmental conditions including iron limitation. H. capsulatum can establish a persistent state within the macrophage. A gap in knowledge exists because the identities and number of proteins regulated by the organism under host conditions has yet to be defined. Lack of such knowledge is an important problem because until these proteins are identified it is unlikely that they can be targeted as new and innovative treatment for histoplasmosis. Results To investigate the proteomic response by H. capsulatum to decreasing iron availability we have created H. capsulatum protein/genomic databases compatible with current mass spectrometric (MS) search engines. Databases were assembled from the H. capsulatum G217B strain genome using gene prediction programs and expressed sequence tag (EST) libraries. Searching these databases with MS data generated from two dimensional (2D) in-gel digestions of proteins resulted in over 50% more proteins identified compared to searching the publicly available fungal databases alone. Using 2D gel electrophoresis combined with statistical analysis we discovered 42 H. capsulatum proteins whose abundance was significantly modulated when iron concentrations were lowered. Altered proteins were identified by mass spectrometry and database searching to be involved in glycolysis, the tricarboxylic acid cycle, lysine metabolism, protein synthesis, and one protein sequence whose function was unknown. Conclusion We have created a bioinformatics platform for H. capsulatum and demonstrated the utility of a proteomic approach by identifying a shift in metabolism the organism utilizes to cope with the hostile conditions provided by the host. We have shown that enzyme transcripts regulated by other fungal pathogens in response to lowering iron availability are also regulated in H. capsulatum at the protein level. We also identified H. capsulatum proteins sensitive to iron level reductions which have yet to be connected to iron availability in other pathogens. These data also indicate the complexity of the response by H. capsulatum to nutritional deprivation. Finally, we demonstrate the importance of a strain specific gene/protein database for H. capsulatum proteomic analysis. PMID:19108728

Simulated linear test applied to quantitative proteomics.

PubMed

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.

Unraveling incompatibility between wheat and the fungal pathogen Zymoseptoria tritici through apoplastic proteomics.

PubMed

Yang, Fen; Li, Wanshun; Derbyshire, Mark; Larsen, Martin R; Rudd, Jason J; Palmisano, Giuseppe

2015-05-08

Hemibiotrophic fungal pathogen Zymoseptoria tritici causes severe foliar disease in wheat. However, current knowledge of molecular mechanisms involved in plant resistance to Z. tritici and Z. tritici virulence factors is far from being complete. The present work investigated the proteome of leaf apoplastic fluid with emphasis on both host wheat and Z. tritici during the compatible and incompatible interactions. The proteomics analysis revealed rapid host responses to the biotrophic growth, including enhanced carbohydrate metabolism, apoplastic defenses and stress, and cell wall reinforcement, might contribute to resistance. Compatibility between the host and the pathogen was associated with inactivated plant apoplastic responses as well as fungal defenses to oxidative stress and perturbation of plant cell wall during the initial biotrophic stage, followed by the strong induction of plant defenses during the necrotrophic stage. To study the role of anti-oxidative stress in Z. tritici pathogenicity in depth, a YAP1 transcription factor regulating antioxidant expression was deleted and showed the contribution to anti-oxidative stress in Z. tritici, but was not required for pathogenicity. This result suggests the functional redundancy of antioxidants in the fungus. The data demonstrate that incompatibility is probably resulted from the proteome-level activation of host apoplastic defenses as well as fungal incapability to adapt to stress and interfere with host cell at the biotrophic stage of the interaction.

Proteomic profiling of mature leaves from oil palm (Elaeis guineensis Jacq.).

PubMed

Tan, Hooi Sin; Jacoby, Richard P; Ong-Abdullah, Meilina; Taylor, Nicolas L; Liddell, Susan; Chee, Wong Wei; Chin, Chiew Foan

2017-04-01

Oil palm is one of the most productive oil bearing crops grown in Southeast Asia. Due to the dwindling availability of agricultural land and increasing demand for high yielding oil palm seedlings, clonal propagation is vital to the oil palm industry. Most commonly, leaf explants are used for in vitro micropropagation of oil palm and to optimize this process it is important to unravel the physiological and molecular mechanisms underlying somatic embryo production from leaves. In this study, a proteomic approach was used to determine protein abundance of mature oil palm leaves. To do this, leaf proteins were extracted using TCA/acetone precipitation protocol and separated by 2DE. A total of 191 protein spots were observed on the 2D gels and 67 of the most abundant protein spots that were consistently observed were selected for further analysis with 35 successfully identified using MALDI TOF/TOF MS. The majority of proteins were classified as being involved in photosynthesis, metabolism, cellular biogenesis, stress response, and transport. This study provides the first proteomic assessment of oil palm leaves in this important oil crop and demonstrates the successful identification of selected proteins spots using the Malaysian Palm Oil Board (MPOB) Elaeis guineensis EST and NCBI-protein databases. The MS data have been deposited in the ProteomeXchange Consortium database with the data set identifier PXD001307. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Combining proteomics and metabolite analyses to unravel cadmium stress-response in poplar leaves.

PubMed

Kieffer, Pol; Planchon, Sébastien; Oufir, Mouhssin; Ziebel, Johanna; Dommes, Jacques; Hoffmann, Lucien; Hausman, Jean-François; Renaut, Jenny

2009-01-01

A proteomic analysis of poplar leaves exposed to cadmium, combined with biochemical analysis of pigments and carbohydrates revealed changes in primary carbon metabolism. Proteomic results suggested that photosynthesis was slightly affected. Together with a growth inhibition, photoassimilates were less needed for developmental processes and could be stored in the form of hexoses or complex sugars, acting also as osmoprotectants. Simultaneously, mitochondrial respiration was upregulated, providing energy needs of cadmium-exposed plants.

Understanding the Mechanism of Thermotolerance Distinct From Heat Shock Response Through Proteomic Analysis of Industrial Strains of Saccharomyces cerevisiae*

PubMed Central

Shui, Wenqing; Xiong, Yun; Xiao, Weidi; Qi, Xianni; Zhang, Yong; Lin, Yuping; Guo, Yufeng; Zhang, Zhidan; Wang, Qinhong; Ma, Yanhe

2015-01-01

Saccharomyces cerevisiae has been intensively studied in responses to different environmental stresses such as heat shock through global omic analysis. However, the S. cerevisiae industrial strains with superior thermotolerance have not been explored in any proteomic studies for elucidating the tolerance mechanism. Recently a new diploid strain was obtained through evolutionary engineering of a parental industrial strain, and it exhibited even higher resistance to prolonged thermal stress. Herein, we performed iTRAQ-based quantitative proteomic analysis on both the parental and evolved industrial strains to further understand the mechanism of thermotolerant adaptation. Out of ∼2600 quantifiable proteins from biological quadruplicates, 193 and 204 proteins were differentially regulated in the parental and evolved strains respectively during heat-stressed growth. The proteomic response of the industrial strains cultivated under prolonged thermal stress turned out to be substantially different from that of the laboratory strain exposed to sudden heat shock. Further analysis of transcription factors underlying the proteomic perturbation also indicated the distinct regulatory mechanism of thermotolerance. Finally, a cochaperone Mdj1 and a metabolic enzyme Adh1 were selected to investigate their roles in mediating heat-stressed growth and ethanol production of yeasts. Our proteomic characterization of the industrial strain led to comprehensive understanding of the molecular basis of thermotolerance, which would facilitate future improvement in the industrially important trait of S. cerevisiae by rational engineering. PMID:25926660

A Comprehensive Guide for Performing Sample Preparation and Top-Down Protein Analysis

PubMed Central

Padula, Matthew P.; Berry, Iain J.; O′Rourke, Matthew B.; Raymond, Benjamin B.A.; Santos, Jerran; Djordjevic, Steven P.

2017-01-01

Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O′Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single ‘spots’ in a polyacrylamide gel, allowing the quantitation of changes in a proteoform′s abundance to ascertain changes in an organism′s phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the ‘Top-Down’. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O’Farrell’s paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism′s proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer. PMID:28387712

A Comprehensive Guide for Performing Sample Preparation and Top-Down Protein Analysis.

PubMed

Padula, Matthew P; Berry, Iain J; O Rourke, Matthew B; Raymond, Benjamin B A; Santos, Jerran; Djordjevic, Steven P

2017-04-07

Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O'Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single 'spots' in a polyacrylamide gel, allowing the quantitation of changes in a proteoform's abundance to ascertain changes in an organism's phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the 'Top-Down'. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O'Farrell's paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism's proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer.

Translational plant proteomics: a perspective.

PubMed

Agrawal, Ganesh Kumar; Pedreschi, Romina; Barkla, Bronwyn J; Bindschedler, Laurence Veronique; Cramer, Rainer; Sarkar, Abhijit; Renaut, Jenny; Job, Dominique; Rakwal, Randeep

2012-08-03

Translational proteomics is an emerging sub-discipline of the proteomics field in the biological sciences. Translational plant proteomics aims to integrate knowledge from basic sciences to translate it into field applications to solve issues related but not limited to the recreational and economic values of plants, food security and safety, and energy sustainability. In this review, we highlight the substantial progress reached in plant proteomics during the past decade which has paved the way for translational plant proteomics. Increasing proteomics knowledge in plants is not limited to model and non-model plants, proteogenomics, crop improvement, and food analysis, safety, and nutrition but to many more potential applications. Given the wealth of information generated and to some extent applied, there is the need for more efficient and broader channels to freely disseminate the information to the scientific community. This article is part of a Special Issue entitled: Translational Proteomics. Copyright © 2012 Elsevier B.V. All rights reserved.

Mass spectrometry based proteomics profiling as diagnostic tool in oncology: current status and future perspective.

PubMed

Findeisen, Peter; Neumaier, Michael

2009-01-01

Proteomics analysis has been heralded as a novel tool for identifying new and specific biomarkers that may improve diagnosis and monitoring of various disease states. Recent years have brought a number of proteomics profiling technologies. Although proteomics profiling has resulted in the detection of disease-associated differences and modification of proteins, current proteomics technologies display certain limitations that are hampering the introduction of these new technologies into clinical laboratory diagnostics and routine applications. In this review, we summarize current advances in mass spectrometry based biomarker discovery. The promises and challenges of this new technology are discussed with particular emphasis on diagnostic perspectives of mass-spectrometry based proteomics profiling for malignant diseases.

Data from quantitative label free proteomics analysis of rat spleen.

PubMed

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.

Highly Reproducible Label Free Quantitative Proteomic Analysis of RNA Polymerase Complexes*

PubMed Central

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:21048197

Dihydrolipoyl dehydrogenase as a potential UVB target in skin epidermis; using an integrated approach of label-free quantitative proteomics and targeted metabolite analysis.

PubMed

Moon, Eunjung; Park, Hye Min; Lee, Choong Hwan; Do, Seon-Gil; Park, Jong-Moon; Han, Na-Young; Do, Moon Ho; Lee, Jong Ha; Lee, Hookeun; Kim, Sun Yeou

2015-03-18

Photodamage is extrinsically induced by overexposure to ultraviolet (UV) radiation, and it increases the risk of various skin disorders. Therefore, discovery of novel biomarkers of photodamage is important. In this study, using LC-MS/MS analysis of epidermis from UVB-irradiated hairless mice, we identified 57 proteins whose levels changed after UVB exposure, and selected 7 proteins related to the tricarboxylic acid (TCA) cycle through pathway analysis. Dihydrolipoyl dehydrogenase (DLD) was the only TCA cycle-associated protein that showed a decreased expression after the UVB exposure. We also performed targeted analysis to detect intermediates and products of the TCA cycle using GC-TOF-MS. Interestingly, malic acid and fumaric acid levels significantly decreased in the UVB-treated group. Our results demonstrate that DLD and its associated metabolites, malic acid and fumaric acid, may be candidate biomarkers of UVB-induced skin photoaging. Additionally, we showed that Aloe vera, a natural skin moisturizer, regulated DLD, malic acid and fumaric acid levels in UVB-exposed epidermis. Our strategy to integrate the proteome and targeted metabolite to detect novel UVB targets will lead to a better understanding of skin photoaging and photodamage. Our study also supports that A. vera exerts significant anti-photodamage activity via regulation of DLD, a novel UVB target, in the epidermis. This study is the first example of an integration of proteomic and metabolite analysis techniques to find new biomarker candidates for the regulation of the UVB-induced skin photoaging. DLD, malic acid, and fumaric acid can be used for development of cosmeceuticals and nutraceuticals regulating the change of skin metabolism induced by the UVB overexposure. Moreover, this is also the first attempt to investigate the role of the TCA cycle in photodamaged epidermis. Our integration of the proteomic and targeted metabolite analyses will lead to a better understanding of the unidentified photobiological results from UVB-irradiated models and can elicit new diagnostic and treatment strategies based on altered metabolism. Copyright © 2015. Published by Elsevier B.V.

Anopheles salivary gland proteomes from major malaria vectors

PubMed Central

2012-01-01

Background Antibody responses against Anopheles salivary proteins can indicate individual exposure to bites of malaria vectors. The extent to which these salivary proteins are species-specific is not entirely resolved. Thus, a better knowledge of the diversity among salivary protein repertoires from various malaria vector species is necessary to select relevant genus-, subgenus- and/or species-specific salivary antigens. Such antigens could be used for quantitative (mosquito density) and qualitative (mosquito species) immunological evaluation of malaria vectors/host contact. In this study, salivary gland protein repertoires (sialomes) from several Anopheles species were compared using in silico analysis and proteomics. The antigenic diversity of salivary gland proteins among different Anopheles species was also examined. Results In silico analysis of secreted salivary gland protein sequences retrieved from an NCBInr database of six Anopheles species belonging to the Cellia subgenus (An. gambiae, An. arabiensis, An. stephensi and An. funestus) and Nyssorhynchus subgenus (An. albimanus and An. darlingi) displayed a higher degree of similarity compared to salivary proteins from closely related Anopheles species. Additionally, computational hierarchical clustering allowed identification of genus-, subgenus- and species-specific salivary proteins. Proteomic and immunoblot analyses performed on salivary gland extracts from four Anopheles species (An. gambiae, An. arabiensis, An. stephensi and An. albimanus) indicated that heterogeneity of the salivary proteome and antigenic proteins was lower among closely related anopheline species and increased with phylogenetic distance. Conclusion This is the first report on the diversity of the salivary protein repertoire among species from the Anopheles genus at the protein level. This work demonstrates that a molecular diversity is exhibited among salivary proteins from closely related species despite their common pharmacological activities. The involvement of these proteins as antigenic candidates for genus-, subgenus- or species-specific immunological evaluation of individual exposure to Anopheles bites is discussed. PMID:23148599

Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation.

PubMed

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.

Comparative proteomic analysis of fibrotic liver of rats fed high fat diet contained lard versus corn oil.

PubMed

Wang, Hualin; Sit, Wat-Hung; Tipoe, George Lim; Liu, Zhiguo; Wan, Jennifer Man-Fan

2017-02-01

The influences of dietary fatty acids on the progress of chronic liver diseases have attracted lots of attentions, but the mechanisms of the effects of lipids rich in saturated fatty acids or PUFAs on hepatic fibrogenesis remain unclear. Female Fischer 344 rats were fed normal chow or chow plus 20% (w/w) of corn oil or lard, respectively, and injected CCl 4 twice a week for 4 weeks to induce liver fibrosis. Masson's staining was adopted to illustrate the fibrosis level. The mRNA expression level of α-SMA and the DNA methylation level of its promoter region were analyzed. A 2-DE gel based proteomic approach was constructed to investigate the differential expression level of hepatic proteome between three diet groups. Histological evaluations and α-SMA expression analysis illustrated the high corn oil intake has no effects on hepatic fibrogenesis, but lard intake aggravated liver fibrosis, partly attributed to DNA demethylation of α-SMA promoter region. 2-DE Gel based proteomic study demonstrated excessive lard consumption elevated the expression of fibrosis related alpha-1-antitrypsin precursor, and endoplasmic reticulum stress related proteins such as heat shock cognate 71 kDa, eukaryotic translation initiation factor 4A1 and protein disulfide isomerase associated 3. Moreover, unlike corn oil rich in PUFAs, lard had no effects to elevate the expression of glutathione S-transferases, but decreased the expression of iron store related proteins heme binding protein 1 and ferritin. Lard intake aggravates CCl 4 induced liver fibrosis via enhancing the expression of fibrogenesis and ER stress related proteins, and disturbing the hepatic transmethylation reaction. Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Platform-independent and Label-free Quantitation of Proteomic Data Using MS1 Extracted Ion Chromatograms in Skyline

PubMed Central

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

Combination of a proteomics approach and reengineering of meso scale network models for prediction of mode-of-action for tyrosine kinase inhibitors.

PubMed

Balabanov, Stefan; Wilhelm, Thomas; Venz, Simone; Keller, Gunhild; Scharf, Christian; Pospisil, Heike; Braig, Melanie; Barett, Christine; Bokemeyer, Carsten; Walther, Reinhard; Brümmendorf, Tim H; Schuppert, Andreas

2013-01-01

In drug discovery, the characterisation of the precise modes of action (MoA) and of unwanted off-target effects of novel molecularly targeted compounds is of highest relevance. Recent approaches for identification of MoA have employed various techniques for modeling of well defined signaling pathways including structural information, changes in phenotypic behavior of cells and gene expression patterns after drug treatment. However, efficient approaches focusing on proteome wide data for the identification of MoA including interference with mutations are underrepresented. As mutations are key drivers of drug resistance in molecularly targeted tumor therapies, efficient analysis and modeling of downstream effects of mutations on drug MoA is a key to efficient development of improved targeted anti-cancer drugs. Here we present a combination of a global proteome analysis, reengineering of network models and integration of apoptosis data used to infer the mode-of-action of various tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) cell lines expressing wild type as well as TKI resistance conferring mutants of BCR-ABL. The inferred network models provide a tool to predict the main MoA of drugs as well as to grouping of drugs with known similar kinase inhibitory activity patterns in comparison to drugs with an additional MoA. We believe that our direct network reconstruction approach, demonstrated on proteomics data, can provide a complementary method to the established network reconstruction approaches for the preclinical modeling of the MoA of various types of targeted drugs in cancer treatment. Hence it may contribute to the more precise prediction of clinically relevant on- and off-target effects of TKIs.

Combination of a Proteomics Approach and Reengineering of Meso Scale Network Models for Prediction of Mode-of-Action for Tyrosine Kinase Inhibitors

PubMed Central

Balabanov, Stefan; Wilhelm, Thomas; Venz, Simone; Keller, Gunhild; Scharf, Christian; Pospisil, Heike; Braig, Melanie; Barett, Christine; Bokemeyer, Carsten; Walther, Reinhard

2013-01-01

In drug discovery, the characterisation of the precise modes of action (MoA) and of unwanted off-target effects of novel molecularly targeted compounds is of highest relevance. Recent approaches for identification of MoA have employed various techniques for modeling of well defined signaling pathways including structural information, changes in phenotypic behavior of cells and gene expression patterns after drug treatment. However, efficient approaches focusing on proteome wide data for the identification of MoA including interference with mutations are underrepresented. As mutations are key drivers of drug resistance in molecularly targeted tumor therapies, efficient analysis and modeling of downstream effects of mutations on drug MoA is a key to efficient development of improved targeted anti-cancer drugs. Here we present a combination of a global proteome analysis, reengineering of network models and integration of apoptosis data used to infer the mode-of-action of various tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) cell lines expressing wild type as well as TKI resistance conferring mutants of BCR-ABL. The inferred network models provide a tool to predict the main MoA of drugs as well as to grouping of drugs with known similar kinase inhibitory activity patterns in comparison to drugs with an additional MoA. We believe that our direct network reconstruction approach, demonstrated on proteomics data, can provide a complementary method to the established network reconstruction approaches for the preclinical modeling of the MoA of various types of targeted drugs in cancer treatment. Hence it may contribute to the more precise prediction of clinically relevant on- and off-target effects of TKIs. PMID:23326482

Proteomic analysis highlights the molecular complexities of native Kv4 channel macromolecular complexes.

PubMed

Marionneau, Céline; Townsend, R Reid; Nerbonne, Jeanne M

2011-04-01

Voltage-gated K(+) (Kv) channels are key determinants of membrane excitability in the nervous and cardiovascular systems, functioning to control resting membrane potentials, shape action potential waveforms and influence the responses to neurotransmitters and neurohormones. Consistent with this functional diversity, multiple types of Kv currents, with distinct biophysical properties and cellular/subcellular distributions, have been identified. Rapidly activating and inactivating Kv currents, typically referred to as I(A) (A-type) in neurons, for example, regulate repetitive firing rates, action potential back-propagation (into dendrites) and modulate synaptic responses. Currents with similar properties, referred to as I(to,f) (fast transient outward), expressed in cardiomyocytes, control the early phase of myocardial action potential repolarization. A number of studies have demonstrated critical roles for pore-forming (α) subunits of the Kv4 subfamily in the generation of native neuronal I(A) and cardiac I(to,f) channels. Studies in heterologous cells have also suggested important roles for a number of Kv channel accessory and regulatory proteins in the generation of functional I(A) and I(to,f) channels. Quantitative mass spectrometry-based proteomic analysis is increasingly recognized as a rapid and, importantly, unbiased, approach to identify the components of native macromolecular protein complexes. The recent application of proteomic approaches to identify the components of native neuronal (and cardiac) Kv4 channel complexes has revealed even greater complexity than anticipated. The continued emphasis on development of improved biochemical and analytical proteomic methods seems certain to accelerate progress and to provide important new insights into the molecular determinants of native ion channel protein complexes. Copyright © 2010 Elsevier Ltd. All rights reserved.

Classification of Complete Proteomes of Different Organisms and Protein Sets Based on Their Protein Distributions in Terms of Some Key Attributes of Proteins

PubMed Central

Ma, Yue; Tuskan, Gerald A.

2018-01-01

The existence of complete genome sequences makes it important to develop different approaches for classification of large-scale data sets and to make extraction of biological insights easier. Here, we propose an approach for classification of complete proteomes/protein sets based on protein distributions on some basic attributes. We demonstrate the usefulness of this approach by determining protein distributions in terms of two attributes: protein lengths and protein intrinsic disorder contents (ID). The protein distributions based on L and ID are surveyed for representative proteome organisms and protein sets from the three domains of life. The two-dimensional maps (designated as fingerprints here) from the protein distribution densities in the LD space defined by ln(L) and ID are then constructed. The fingerprints for different organisms and protein sets are found to be distinct with each other, and they can therefore be used for comparative studies. As a test case, phylogenetic trees have been constructed based on the protein distribution densities in the fingerprints of proteomes of organisms without performing any protein sequence comparison and alignments. The phylogenetic trees generated are biologically meaningful, demonstrating that the protein distributions in the LD space may serve as unique phylogenetic signals of the organisms at the proteome level. PMID:29686995

Proteomics of Dense Core Secretory Vesicles Reveal Distinct Protein Categories for Secretion of Neuroeffectors for Cell-Cell Communication

PubMed Central

Wegrzyn, Jill L.; Bark, Steven J.; Funkelstein, Lydiane; Mosier, Charles; Yap, Angel; Kazemi-Esfarjani, Parasa; La Spada, Albert; Sigurdson, Christina; O’Connor, Daniel T.; Hook, Vivian

2010-01-01

Regulated secretion of neurotransmitters and neurohumoural factors from dense core secretory vesicles provides essential neuroeffectors for cell-cell communication in the nervous and endocrine systems. This study provides comprehensive proteomic characterization of the categories of proteins in chromaffin dense core secretory vesicles that participate in cell-cell communication from the adrenal medulla. Proteomic studies were conducted by nano-HPLC Chip MS/MS tandem mass spectrometry. Results demonstrate that these secretory vesicles contain proteins of distinct functional categories consisting of neuropeptides and neurohumoural factors, protease systems, neurotransmitter enzymes and transporters, receptors, enzymes for biochemical processes, reduction/oxidation regulation, ATPases, protein folding, lipid biochemistry, signal transduction, exocytosis, calcium regulation, as well as structural and cell adhesion proteins. The secretory vesicle proteomic data identified 371 distinct proteins in the soluble fraction and 384 distinct membrane proteins, for a total of 686 distinct secretory vesicle proteins. Notably, these proteomic analyses illustrate the presence of several neurological disease-related proteins in these secretory vesicles, including huntingtin interacting protein, cystatin C, ataxin 7, and prion protein. Overall, these findings demonstrate that multiple protein categories participate in dense core secretory vesicles for production, storage, and secretion of bioactive neuroeffectors for cell-cell communication in health and disease. PMID:20695487

Systematic Evaluation of the Use of Human Plasma and Serum for Mass-Spectrometry-Based Shotgun Proteomics.

PubMed

Lan, Jiayi; Núñez Galindo, Antonio; Doecke, James; Fowler, Christopher; Martins, Ralph N; Rainey-Smith, Stephanie R; Cominetti, Ornella; Dayon, Loïc

2018-04-06

Over the last two decades, EDTA-plasma has been used as the preferred sample matrix for human blood proteomic profiling. Serum has also been employed widely. Only a few studies have assessed the difference and relevance of the proteome profiles obtained from plasma samples, such as EDTA-plasma or lithium-heparin-plasma, and serum. A more complete evaluation of the use of EDTA-plasma, heparin-plasma, and serum would greatly expand the comprehensiveness of shotgun proteomics of blood samples. In this study, we evaluated the use of heparin-plasma with respect to EDTA-plasma and serum to profile blood proteomes using a scalable automated proteomic pipeline (ASAP 2 ). The use of plasma and serum for mass-spectrometry-based shotgun proteomics was first tested with commercial pooled samples. The proteome coverage consistency and the quantitative performance were compared. Furthermore, protein measurements in EDTA-plasma and heparin-plasma samples were comparatively studied using matched sample pairs from 20 individuals from the Australian Imaging, Biomarkers and Lifestyle (AIBL) Study. We identified 442 proteins in common between EDTA-plasma and heparin-plasma samples. Overall agreement of the relative protein quantification between the sample pairs demonstrated that shotgun proteomics using workflows such as the ASAP 2 is suitable in analyzing heparin-plasma and that such sample type may be considered in large-scale clinical research studies. Moreover, the partial proteome coverage overlaps (e.g., ∼70%) showed that measures from heparin-plasma could be complementary to those obtained from EDTA-plasma.

Affinity Proteomics in the mountains: Alpbach 2015.

PubMed

Taussig, Michael J

2016-09-25

The 2015 Alpbach Workshop on Affinity Proteomics, organised by the EU AFFINOMICS consortium, was the 7th workshop in this series. As in previous years, the focus of the event was the current state of affinity methods for proteome analysis, including complementarity with mass spectrometry, progress in recombinant binder production methods, alternatives to classical antibodies as affinity reagents, analysis of proteome targets, industry focus on biomarkers, and diagnostic and clinical applications. The combination of excellent science with Austrian mountain scenery and winter sports engender an atmosphere that makes this series of workshops exceptional. The articles in this Special Issue represent a cross-section of the presentations at the 2015 meeting. Copyright © 2016 Elsevier B.V. All rights reserved.

Sherlock Holmes and the proteome--a detective story.

PubMed

Righetti, Pier Giorgio; Boschetti, Egisto

2007-02-01

The performance of a hexapeptide ligand library in capturing the 'hidden proteome' is illustrated and evaluated. This library, insolubilized on an organic polymer and available under the trade name 'Equalizer Bead Technology', acts by capturing all components of a given proteome, by concentrating rare and very rare proteins, and simultaneously diluting the abundant ones. This results in a proteome of 'normalized' relative abundances, amenable to analysis by MS and any other analytical tool. Examples are given of analysis of human urine and serum, as well as cell and tissue lysates, such as Escherichia coli and Saccharomyces cerevisiae extracts. Another important application is impurity tracking and polishing of recombinant DNA products, especially biopharmaceuticals meant for human consumption.

A computational pipeline for the development of multi-marker bio-signature panels and ensemble classifiers

PubMed Central

2012-01-01

Background Biomarker panels derived separately from genomic and proteomic data and with a variety of computational methods have demonstrated promising classification performance in various diseases. An open question is how to create effective proteo-genomic panels. The framework of ensemble classifiers has been applied successfully in various analytical domains to combine classifiers so that the performance of the ensemble exceeds the performance of individual classifiers. Using blood-based diagnosis of acute renal allograft rejection as a case study, we address the following question in this paper: Can acute rejection classification performance be improved by combining individual genomic and proteomic classifiers in an ensemble? Results The first part of the paper presents a computational biomarker development pipeline for genomic and proteomic data. The pipeline begins with data acquisition (e.g., from bio-samples to microarray data), quality control, statistical analysis and mining of the data, and finally various forms of validation. The pipeline ensures that the various classifiers to be combined later in an ensemble are diverse and adequate for clinical use. Five mRNA genomic and five proteomic classifiers were developed independently using single time-point blood samples from 11 acute-rejection and 22 non-rejection renal transplant patients. The second part of the paper examines five ensembles ranging in size from two to 10 individual classifiers. Performance of ensembles is characterized by area under the curve (AUC), sensitivity, and specificity, as derived from the probability of acute rejection for individual classifiers in the ensemble in combination with one of two aggregation methods: (1) Average Probability or (2) Vote Threshold. One ensemble demonstrated superior performance and was able to improve sensitivity and AUC beyond the best values observed for any of the individual classifiers in the ensemble, while staying within the range of observed specificity. The Vote Threshold aggregation method achieved improved sensitivity for all 5 ensembles, but typically at the cost of decreased specificity. Conclusion Proteo-genomic biomarker ensemble classifiers show promise in the diagnosis of acute renal allograft rejection and can improve classification performance beyond that of individual genomic or proteomic classifiers alone. Validation of our results in an international multicenter study is currently underway. PMID:23216969

A computational pipeline for the development of multi-marker bio-signature panels and ensemble classifiers.

PubMed

Günther, Oliver P; Chen, Virginia; Freue, Gabriela Cohen; Balshaw, Robert F; Tebbutt, Scott J; Hollander, Zsuzsanna; Takhar, Mandeep; McMaster, W Robert; McManus, Bruce M; Keown, Paul A; Ng, Raymond T

2012-12-08

Biomarker panels derived separately from genomic and proteomic data and with a variety of computational methods have demonstrated promising classification performance in various diseases. An open question is how to create effective proteo-genomic panels. The framework of ensemble classifiers has been applied successfully in various analytical domains to combine classifiers so that the performance of the ensemble exceeds the performance of individual classifiers. Using blood-based diagnosis of acute renal allograft rejection as a case study, we address the following question in this paper: Can acute rejection classification performance be improved by combining individual genomic and proteomic classifiers in an ensemble? The first part of the paper presents a computational biomarker development pipeline for genomic and proteomic data. The pipeline begins with data acquisition (e.g., from bio-samples to microarray data), quality control, statistical analysis and mining of the data, and finally various forms of validation. The pipeline ensures that the various classifiers to be combined later in an ensemble are diverse and adequate for clinical use. Five mRNA genomic and five proteomic classifiers were developed independently using single time-point blood samples from 11 acute-rejection and 22 non-rejection renal transplant patients. The second part of the paper examines five ensembles ranging in size from two to 10 individual classifiers. Performance of ensembles is characterized by area under the curve (AUC), sensitivity, and specificity, as derived from the probability of acute rejection for individual classifiers in the ensemble in combination with one of two aggregation methods: (1) Average Probability or (2) Vote Threshold. One ensemble demonstrated superior performance and was able to improve sensitivity and AUC beyond the best values observed for any of the individual classifiers in the ensemble, while staying within the range of observed specificity. The Vote Threshold aggregation method achieved improved sensitivity for all 5 ensembles, but typically at the cost of decreased specificity. Proteo-genomic biomarker ensemble classifiers show promise in the diagnosis of acute renal allograft rejection and can improve classification performance beyond that of individual genomic or proteomic classifiers alone. Validation of our results in an international multicenter study is currently underway.

Comparison of methods for profiling O-glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi-institutional study of IgA1.

PubMed

Wada, Yoshinao; Dell, Anne; Haslam, Stuart M; Tissot, Bérangère; Canis, Kévin; Azadi, Parastoo; Bäckström, Malin; Costello, Catherine E; Hansson, Gunnar C; Hiki, Yoshiyuki; Ishihara, Mayumi; Ito, Hiromi; Kakehi, Kazuaki; Karlsson, Niclas; Hayes, Catherine E; Kato, Koichi; Kawasaki, Nana; Khoo, Kay-Hooi; Kobayashi, Kunihiko; Kolarich, Daniel; Kondo, Akihiro; Lebrilla, Carlito; Nakano, Miyako; Narimatsu, Hisashi; Novak, Jan; Novotny, Milos V; Ohno, Erina; Packer, Nicolle H; Palaima, Elizabeth; Renfrow, Matthew B; Tajiri, Michiko; Thomsson, Kristina A; Yagi, Hirokazu; Yu, Shin-Yi; Taniguchi, Naoyuki

2010-04-01

The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiative's activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.

Proteomic analysis of three gonad types of swamp eel reveals genes differentially expressed during sex reversal.

PubMed

Sheng, Yue; Zhao, Wei; Song, Ying; Li, Zhigang; Luo, Majing; Lei, Quan; Cheng, Hanhua; Zhou, Rongjia

2015-05-18

A variety of mechanisms are engaged in sex determination in vertebrates. The teleost fish swamp eel undergoes sex reversal naturally and is an ideal model for vertebrate sexual development. However, the importance of proteome-wide scanning for gonad reversal was not previously determined. We report a 2-D electrophoresis analysis of three gonad types of proteomes during sex reversal. MS/MS analysis revealed a group of differentially expressed proteins during ovary to ovotestis to testis transformation. Cbx3 is up-regulated during gonad reversal and is likely to have a role in spermatogenesis. Rab37 is down-regulated during the reversal and is mainly associated with oogenesis. Both Cbx3 and Rab37 are linked up in a protein network. These datasets in gonadal proteomes provide a new resource for further studies in gonadal development.

Analysis of PNGase F-resistant N-glycopeptides using SugarQb for Proteome Discoverer 2.1 reveals cryptic substrate specificities.

PubMed

Stadlmann, Johannes; Hoi, David M; Taubenschmid, Jasmin; Mechtler, Karl; Penninger, Josef M

2018-05-18

SugarQb (www.imba.oeaw.ac.at/sugarqb) is a freely available collection of computational tools for the automated identification of intact glycopeptides from high-resolution HCD MS/MS data-sets in the Proteome Discoverer environment. We report the migration of SugarQb to the latest and free version of Proteome Discoverer 2.1, and apply it to the analysis of PNGase F-resistant N-glycopeptides from mouse embryonic stem cells. The analysis of intact glycopeptides highlights unexpected technical limitations to PNGase F-dependent glycoproteomic workflows at the proteome level, and warrants a critical re-interpretation of seminal data-sets in the context of N-glycosylation-site prediction. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes

PubMed Central

Zhang, Yanling; Zhang, Yong; Adachi, Jun; Olsen, Jesper V.; Shi, Rong; de Souza, Gustavo; Pasini, Erica; Foster, Leonard J.; Macek, Boris; Zougman, Alexandre; Kumar, Chanchal; Wiśniewski, Jacek R.; Jun, Wang; Mann, Matthias

2007-01-01

Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS and stringent validation criteria, false positive identification rates in MAPU are lower than 1:1000. Thus MAPU datasets can serve as reference proteomes in biomarker discovery. MAPU contains the peptides identifying each protein, measured masses, scores and intensities and is freely available at using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic annotation information and links to other public databases are provided in MAPU and we plan to add further analysis tools. PMID:17090601

Proteomics of filamentous fungi.

PubMed

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.

Astragaloside IV Attenuates Glutamate-Induced Neurotoxicity in PC12 Cells through Raf-MEK-ERK Pathway.

PubMed

Yue, Rongcai; Li, Xia; Chen, Bingyang; Zhao, Jing; He, Weiwei; Yuan, Hu; Yuan, Xing; Gao, Na; Wu, Guozhen; Jin, Huizi; Shan, Lei; Zhang, Weidong

2015-01-01

Astragaloside IV (AGS-IV) is a main active ingredient of Astragalus membranaceus Bunge, a medicinal herb prescribed as an immunostimulant, hepatoprotective, antiperspirant, a diuretic or a tonic as documented in Chinese Materia Medica. In the present study, we employed a high-throughput comparative proteomic approach based on 2D-nano-LC-MS/MS to investigate the possible mechanism of action involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. Differential proteins were identified, among which 13 proteins survived the stringent filter criteria and were further included for functional discussion. Two proteins (vimentin and Gap43) were randomly selected, and their expression levels were further confirmed by western blots analysis. The results matched well with those of proteomics. Furthermore, network analysis of protein-protein interactions (PPI) and pathways enrichment with AGS-IV associated proteins were carried out to illustrate its underlying molecular mechanism. Proteins associated with signal transduction, immune system, signaling molecules and interaction, and energy metabolism play important roles in neuroprotective effect of AGS-IV and Raf-MEK-ERK pathway was involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. This study demonstrates that comparative proteomics based on shotgun approach is a valuable tool for molecular mechanism studies, since it allows the simultaneously evaluate the global proteins alterations.

Vascular Proteomics Reveal Novel Proteins Involved in SMC Phenotypic Change: OLR1 as a SMC Receptor Regulating Proliferation and Inflammatory Response

PubMed Central

Kang, Dong Hoon; Choi, Mina; Chang, Soyoung; Lee, Min Young; Lee, Doo Jae; Choi, Kyungsun; Park, Junseong; Han, Eun Chun; Hwang, Daehee; Kwon, Kihwan; Jo, Hanjoong; Choi, Chulhee; Kang, Sang Won

2015-01-01

Neointimal hyperplasia of vascular smooth muscle cells (VSMC) plays a critical role in atherosclerotic plaque formation and in-stent restenosis, but the underlying mechanisms are still incompletely understood. We performed a proteomics study to identify novel signaling molecules organizing the VSMC hyperplasia. The differential proteomics analysis in a balloon-induced injury model of rat carotid artery revealed that the expressions of 44 proteins are changed within 3 days post injury. The combination of cellular function assays and a protein network analysis further demonstrated that 27 out of 44 proteins constitute key signaling networks orchestrating the phenotypic change of VSMC from contractile to epithelial-like synthetic. Among the list of proteins, the in vivo validation specifically revealed that six proteins (Rab15, ITR, OLR1, PDHβ, PTPε) are positive regulators for VSMC hyperplasia. In particular, the OLR1 played dual roles in the VSMC hyperplasia by directly mediating oxidized LDL-induced monocyte adhesion via NF-κB activation and by assisting the PDGF-induced proliferation/migration. Importantly, OLR1 and PDGFRβ were associated in close proximity in the plasma membrane. Thus, this study elicits the protein network organizing the phenotypic change of VSMC in the vascular injury diseases such as atherosclerosis and discovers OLR1 as a novel molecular link between the proliferative and inflammatory responses of VSMCs. PMID:26305474

Surface modified capillary electrophoresis combined with in solution isoelectric focusing and MALDI-TOF/TOF MS: a gel-free multidimensional electrophoresis approach for proteomic profiling--exemplified on human follicular fluid.

PubMed

Hanrieder, Jörg; Zuberovic, Aida; Bergquist, Jonas

2009-04-24

Development of miniaturized analytical tools continues to be of great interest to face the challenges in proteomic analysis of complex biological samples such as human body fluids. In the light of these challenges, special emphasis is put on the speed and simplicity of newly designed technological approaches as well as the need for cost efficiency and low sample consumption. In this study, we present an alternative multidimensional bottom-up approach for proteomic profiling for fast, efficient and sensitive protein analysis in complex biological matrices. The presented setup was based on sample pre-fractionation using microscale in solution isoelectric focusing (IEF) followed by tryptic digestion and subsequent capillary electrophoresis (CE) coupled off-line to matrix assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI TOF MS/MS). For high performance CE-separation, PolyE-323 modified capillaries were applied to minimize analyte-wall interactions. The potential of the analytical setup was demonstrated on human follicular fluid (hFF) representing a typical complex human body fluid with clinical implication. The obtained results show significant identification of 73 unique proteins (identified at 95% significance level), including mostly acute phase proteins but also protein identities that are well known to be extensively involved in follicular development.

Medullospheres from DAOY, UW228 and ONS-76 cells: increased stem cell population and proteomic modifications.

PubMed

Zanini, Cristina; Ercole, Elisabetta; Mandili, Giorgia; Salaroli, Roberta; Poli, Alice; Renna, Cristiano; Papa, Valentina; Cenacchi, Giovanna; Forni, Marco

2013-01-01

Medulloblastoma (MB) is an aggressive pediatric tumor of the Central Nervous System (CNS) usually treated according to a refined risk stratification. The study of cancer stem cells (CSC) in MB is a promising approach aimed at finding new treatment strategies. The CSC compartment was studied in three characterized MB cell lines (DAOY, UW228 and ONS-76) grown in standard adhesion as well as being grown as spheres, which enables expansion of the CSC population. MB cell lines, grown in adherence and as spheres, were subjected to morphologic analysis at the light and electron microscopic level, as well as cytofluorimetric determinations. Medullospheres (MBS) were shown to express increasingly immature features, along with the stem cells markers: CD133, Nestin and β-catenin. Proteomic analysis highlighted the differences between MB cell lines, demonstrating a unique protein profile for each cell line, and minor differences when grown as spheres. In MBS, MALDI-TOF also identified some proteins, that have been linked to tumor progression and resistance, such as Nucleophosmin (NPM). In addition, immunocytochemistry detected Sox-2 as a stemness marker of MBS, as well as confirming high NPM expression. Culture conditioning based on low attachment flasks and specialized medium may provide new data on the staminal compartment of CNS tumors, although a proteomic profile of CSC is still elusive for MB.

Medullospheres from DAOY, UW228 and ONS-76 Cells: Increased Stem Cell Population and Proteomic Modifications

PubMed Central

Zanini, Cristina; Ercole, Elisabetta; Mandili, Giorgia; Salaroli, Roberta; Poli, Alice; Renna, Cristiano; Papa, Valentina; Cenacchi, Giovanna; Forni, Marco

2013-01-01

Background Medulloblastoma (MB) is an aggressive pediatric tumor of the Central Nervous System (CNS) usually treated according to a refined risk stratification. The study of cancer stem cells (CSC) in MB is a promising approach aimed at finding new treatment strategies. Methodology/Principal Findings The CSC compartment was studied in three characterized MB cell lines (DAOY, UW228 and ONS-76) grown in standard adhesion as well as being grown as spheres, which enables expansion of the CSC population. MB cell lines, grown in adherence and as spheres, were subjected to morphologic analysis at the light and electron microscopic level, as well as cytofluorimetric determinations. Medullospheres (MBS) were shown to express increasingly immature features, along with the stem cells markers: CD133, Nestin and β-catenin. Proteomic analysis highlighted the differences between MB cell lines, demonstrating a unique protein profile for each cell line, and minor differences when grown as spheres. In MBS, MALDI-TOF also identified some proteins, that have been linked to tumor progression and resistance, such as Nucleophosmin (NPM). In addition, immunocytochemistry detected Sox-2 as a stemness marker of MBS, as well as confirming high NPM expression. Conclusions/Significance Culture conditioning based on low attachment flasks and specialized medium may provide new data on the staminal compartment of CNS tumors, although a proteomic profile of CSC is still elusive for MB. PMID:23717474

Analysis of the Aspergillus fumigatus proteome reveals metabolic changes and the activation of the pseurotin A biosynthesis gene cluster in response to hypoxia.

PubMed

Vödisch, Martin; Scherlach, Kirstin; Winkler, Robert; Hertweck, Christian; Braun, Hans-Peter; Roth, Martin; Haas, Hubertus; Werner, Ernst R; Brakhage, Axel A; Kniemeyer, Olaf

2011-05-06

The mold Aspergillus fumigatus is the most important airborne fungal pathogen. Adaptation to hypoxia represents an important virulence attribute for A. fumigatus. Therefore, we aimed at obtaining a comprehensive overview about this process on the proteome level. To ensure highly reproducible growth conditions, an oxygen-controlled, glucose-limited chemostat cultivation was established. Two-dimensional gel electrophoresis analysis of mycelial and mitochondrial proteins as well as two-dimensional Blue Native/SDS-gel separation of mitochondrial membrane proteins led to the identification of 117 proteins with an altered abundance under hypoxic in comparison to normoxic conditions. Hypoxia induced an increased activity of glycolysis, the TCA-cycle, respiration, and amino acid metabolism. Consistently, the cellular contents in heme, iron, copper, and zinc increased. Furthermore, hypoxia induced biosynthesis of the secondary metabolite pseurotin A as demonstrated at proteomic, transcriptional, and metabolite levels. The observed and so far not reported stimulation of the biosynthesis of a secondary metabolite by oxygen depletion may also affect the survival of A. fumigatus in hypoxic niches of the human host. Among the proteins so far not implicated in hypoxia adaptation, an NO-detoxifying flavohemoprotein was one of the most highly up-regulated proteins which indicates a link between hypoxia and the generation of nitrosative stress in A. fumigatus.

Proteomic analysis of polyribosomes identifies splicing factors as potential regulators of translation during mitosis

PubMed Central

Hofmann, Sarah; Elman, Tamar; Shenoy, Anjana; Geiger, Tamar; Elkon, Ran; Ehrlich, Marcelo

2017-01-01

Abstract Precise regulation of mRNA translation is critical for proper cell division, but little is known about the factors that mediate it. To identify mRNA-binding proteins that regulate translation during mitosis, we analyzed the composition of polysomes from interphase and mitotic cells using unbiased quantitative mass-spectrometry (LC–MS/MS). We found that mitotic polysomes are enriched with a subset of proteins involved in RNA processing, including alternative splicing and RNA export. To demonstrate that these may indeed be regulators of translation, we focused on heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a test case and confirmed that it is recruited to elongating ribosomes during mitosis. Then, using a combination of pulsed SILAC, metabolic labeling and ribosome profiling, we showed that knockdown of hnRNP C affects both global and transcript-specific translation rates and found that hnRNP C is specifically important for translation of mRNAs that encode ribosomal proteins and translation factors. Taken together, our results demonstrate how proteomic analysis of polysomes can provide insight into translation regulation under various cellular conditions of interest and suggest that hnRNP C facilitates production of translation machinery components during mitosis to provide daughter cells with the ability to efficiently synthesize proteins as they enter G1 phase. PMID:28460002

Pseudomonas aeruginosa cells attached to a surface display a typical proteome early as 20 minutes of incubation

PubMed Central

Crouzet, Marc; Claverol, Stéphane; Lomenech, Anne-Marie; Le Sénéchal, Caroline; Costaglioli, Patricia; Barthe, Christophe; Garbay, Bertrand; Bonneu, Marc

2017-01-01

Biofilms are present in all environments and often result in negative effects due to properties of the biofilm lifestyle and especially antibiotics resistance. Biofilms are associated with chronic infections. Controlling bacterial attachment, the first step of biofilm formation, is crucial for fighting against biofilm and subsequently preventing the persistence of infection. Thus deciphering the underlying molecular mechanisms involved in attachment could allow discovering molecular targets from it would be possible to develop inhibitors against bacterial colonization and potentiate antibiotherapy. To identify the key components and pathways that aid the opportunistic pathogen Pseudomonas aeruginosa in attachment we performed for the first time a proteomic analysis as early as after 20 minutes of incubation using glass wool fibers as a surface. We compared the protein contents of the attached and unattached bacteria. Using mass spectrometry, 3043 proteins were identified. Our results showed that, as of 20 minutes of incubation, using stringent quantification criteria 616 proteins presented a modification of their abundance in the attached cells compared to their unattached counterparts. The attached cells presented an overall reduced gene expression and characteristics of slow-growing cells. The over-accumulation of outer membrane proteins, periplasmic folding proteins and O-antigen chain length regulators was also observed, indicating a profound modification of the cell envelope. Consistently the sigma factor AlgU required for cell envelope homeostasis was highly over-accumulated in attached cells. In addition our data suggested a role of alarmone (p)ppGpp and polyphosphate during the early attachment phase. Furthermore, almost 150 proteins of unknown function were differentially accumulated in the attached cells. Our proteomic analysis revealed the existence of distinctive biological features in attached cells as early as 20 minutes of incubation. Analysis of some mutants demonstrated the interest of this proteomic approach in identifying genes involved in the early phase of adhesion to a surface. PMID:28678862

A knowledge-based T2-statistic to perform pathway analysis for quantitative proteomic data

PubMed Central

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://github.com/roqe/T2GA. PMID:28622336

A knowledge-based T2-statistic to perform pathway analysis for quantitative proteomic data.

PubMed

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://github.com/roqe/T2GA.

An Informatics-assisted Label-free Approach for Personalized Tissue Membrane Proteomics: Case Study on Colorectal Cancer*

PubMed Central

Han, Chia-Li; Chen, Jinn-Shiun; Chan, Err-Cheng; Wu, Chien-Peng; Yu, Kun-Hsing; Chen, Kuei-Tien; Tsou, Chih-Chiang; Tsai, Chia-Feng; Chien, Chih-Wei; Kuo, Yung-Bin; Lin, Pei-Yi; Yu, Jau-Song; Hsueh, Chuen; Chen, Min-Chi; Chan, Chung-Chuan; Chang, Yu-Sun; Chen, Yu-Ju

2011-01-01

We developed a multiplexed label-free quantification strategy, which integrates an efficient gel-assisted digestion protocol, high-performance liquid chromatography tandem MS analysis, and a bioinformatics alignment method to determine personalized proteomic profiles for membrane proteins in human tissues. This strategy provided accurate (6% error) and reproducible (34% relative S.D.) quantification of three independently purified membrane fractions from the same human colorectal cancer (CRC) tissue. Using CRC as a model, we constructed the personalized membrane protein atlas of paired tumor and adjacent normal tissues from 28 patients with different stages of CRC. Without fractionation, this strategy confidently quantified 856 proteins (≥2 unique peptides) across different patients, including the first and robust detection (Mascot score: 22,074) of the well-documented CRC marker, carcinoembryonic antigen 5 by a discovery-type proteomics approach. Further validation of a panel of proteins, annexin A4, neutrophils defensin A1, and claudin 3, confirmed differential expression levels and high occurrences (48–70%) in 60 CRC patients. The most significant discovery is the overexpression of stomatin-like 2 (STOML2) for early diagnostic and prognostic potential. Increased expression of STOML2 was associated with decreased CRC-related survival; the mean survival period was 34.77 ± 2.03 months in patients with high STOML2 expression, whereas 53.67 ± 3.46 months was obtained for patients with low STOML2 expression. Further analysis by ELISA verified that plasma concentrations of STOML2 in early-stage CRC patients were elevated as compared with those of healthy individuals (p < 0.001), suggesting that STOML2 may be a noninvasive serological biomarker for early CRC diagnosis. The overall sensitivity of STOML2 for CRC detection was 71%, which increased to 87% when combined with CEA measurements. This study demonstrated a sensitive, label-free strategy for differential analysis of tissue membrane proteome, which may provide a roadmap for the subsequent identification of molecular target candidates of multiple cancer types. PMID:21209152

Comparative Testis Tissue Proteomics Using 2-Dye Versus 3-Dye DIGE Analysis.

PubMed

Holland, Ashling

2018-01-01

Comparative tissue proteomics aims to analyze alterations of the proteome in response to a stimulus. Two-dimensional difference gel electrophoresis (2D-DIGE) is a modified and advanced form of 2D gel electrophoresis. DIGE is a powerful biochemical method that compares two or three protein samples on the same analytical gel, and can be used to establish differentially expressed protein levels between healthy normal and diseased pathological tissue sample groups. Minimal DIGE labeling can be used via a 2-dye system with Cy3 and Cy5 or a 3-dye system with Cy2, Cy3, and Cy5 to fluorescently label samples with CyDye flours pre-electrophoresis. DIGE circumvents gel-to-gel variability by multiplexing samples to a single gel and through the use of a pooled internal standard for normalization. This form of quantitative high-resolution proteomics facilitates the comparative analysis and evaluation of tissue protein compositions. Comparing tissue groups under different conditions is crucially important for advancing the biomedical field by characterization of cellular processes, understanding pathophysiological development and tissue biomarker discovery. This chapter discusses 2D-DIGE as a comparative tissue proteomic technique and describes in detail the experimental steps required for comparative proteomic analysis employing both options of 2-dye and 3-dye DIGE minimal labeling.

Integrating Mass Spectrometry of Intact Protein Complexes into Structural Proteomics

PubMed Central

Hyung, Suk-Joon; Ruotolo, Brandon T.

2013-01-01

Summary Mass spectrometry analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other mass spectrometry approaches in structural biology. Methods such as H/D exchange, oxidative foot-printing, chemical cross-linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative mass spectrometry approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly-advancing area. PMID:22611037

Challenges and Strategies for Proteome Analysis of the Interaction of Human Pathogenic Fungi with Host Immune Cells.

PubMed

Krüger, Thomas; Luo, Ting; Schmidt, Hella; Shopova, Iordana; Kniemeyer, Olaf

2015-12-14

Opportunistic human pathogenic fungi including the saprotrophic mold Aspergillus fumigatus and the human commensal Candida albicans can cause severe fungal infections in immunocompromised or critically ill patients. The first line of defense against opportunistic fungal pathogens is the innate immune system. Phagocytes such as macrophages, neutrophils and dendritic cells are an important pillar of the innate immune response and have evolved versatile defense strategies against microbial pathogens. On the other hand, human-pathogenic fungi have sophisticated virulence strategies to counteract the innate immune defense. In this context, proteomic approaches can provide deeper insights into the molecular mechanisms of the interaction of host immune cells with fungal pathogens. This is crucial for the identification of both diagnostic biomarkers for fungal infections and therapeutic targets. Studying host-fungal interactions at the protein level is a challenging endeavor, yet there are few studies that have been undertaken. This review draws attention to proteomic techniques and their application to fungal pathogens and to challenges, difficulties, and limitations that may arise in the course of simultaneous dual proteome analysis of host immune cells interacting with diverse morphotypes of fungal pathogens. On this basis, we discuss strategies to overcome these multifaceted experimental and analytical challenges including the viability of immune cells during co-cultivation, the increased and heterogeneous protein complexity of the host proteome dynamically interacting with the fungal proteome, and the demands on normalization strategies in terms of relative quantitative proteome analysis.

Quantitative proteomic analysis of paired colorectal cancer and non-tumorigenic tissues reveals signature proteins and perturbed pathways involved in CRC progression and metastasis.

PubMed

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.

An FD-LC-MS/MS Proteomic Strategy for Revealing Cellular Protein Networks: A Conditional Superoxide Dismutase 1 Knockout Cells

PubMed Central

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

Analysis of the variability of human normal urine by 2D-GE reveals a "public" and a "private" proteome.

PubMed

Molina, Laurence; Salvetat, Nicolas; Ameur, Randa Ben; Peres, Sabine; Sommerer, Nicolas; Jarraya, Fayçal; Ayadi, Hammadi; Molina, Franck; Granier, Claude

2011-12-10

The characterization of the normal urinary proteome is steadily progressing and represents a major interest in the assessment of clinical urinary biomarkers. To estimate quantitatively the variability of the normal urinary proteome, urines of 20 healthy people were collected. We first evaluated the impact of the sample conservation temperature on urine proteome integrity. Keeping the urine sample at RT or at +4°C until storage at -80°C seems the best way for long-term storage of samples for 2D-GE analysis. The quantitative variability of the normal urinary proteome was estimated on the 20 urines mapped by 2D-GE. The occurrence of the 910 identified spots was analysed throughout the gels and represented in a virtual 2D gel. Sixteen percent of the spots were found to occur in all samples and 23% occurred in at least 90% of urines. About 13% of the protein spots were present only in 10% or less of the samples, thus representing the most variable part of the normal urinary proteome. Twenty proteins corresponding to a fraction of the fully conserved spots were identified by mass spectrometry. In conclusion, a "public" urinary proteome, common to healthy individuals, seems to coexist with a "private" urinary proteome, which is more specific to each individual. Copyright © 2011 Elsevier B.V. All rights reserved.

Transformative Impact of Proteomics on Cardiovascular Health and Disease: A Scientific Statement From the American Heart Association.

PubMed

Lindsey, Merry L; Mayr, Manuel; Gomes, Aldrin V; Delles, Christian; Arrell, D Kent; Murphy, Anne M; Lange, Richard A; Costello, Catherine E; Jin, Yu-Fang; Laskowitz, Daniel T; Sam, Flora; Terzic, Andre; Van Eyk, Jennifer; Srinivas, Pothur R

2015-09-01

The year 2014 marked the 20th anniversary of the coining of the term proteomics. The purpose of this scientific statement is to summarize advances over this period that have catalyzed our capacity to address the experimental, translational, and clinical implications of proteomics as applied to cardiovascular health and disease and to evaluate the current status of the field. Key successes that have energized the field are delineated; opportunities for proteomics to drive basic science research, facilitate clinical translation, and establish diagnostic and therapeutic healthcare algorithms are discussed; and challenges that remain to be solved before proteomic technologies can be readily translated from scientific discoveries to meaningful advances in cardiovascular care are addressed. Proteomics is the result of disruptive technologies, namely, mass spectrometry and database searching, which drove protein analysis from 1 protein at a time to protein mixture analyses that enable large-scale analysis of proteins and facilitate paradigm shifts in biological concepts that address important clinical questions. Over the past 20 years, the field of proteomics has matured, yet it is still developing rapidly. The scope of this statement will extend beyond the reaches of a typical review article and offer guidance on the use of next-generation proteomics for future scientific discovery in the basic research laboratory and clinical settings. © 2015 American Heart Association, Inc.

Profiling modifications for glioblastoma proteome using ultra-tolerant database search: Are the peptide mass shifts biologically relevant or chemically induced?

PubMed

Tarasova, Irina A; Chumakov, Peter M; Moshkovskii, Sergei A; Gorshkov, Mikhail V

2018-05-17

Peptide mass shifts were profiled using ultra-tolerant database search strategy for shotgun proteomics data sets of human glioblastoma cell lines demonstrating strong response to the type I interferon (IFNα-2b) treatment. The main objective of this profiling was revealing the cell response to IFN treatment at the level of protein modifications. To achieve this objective, statistically significant changes in peptide mass shift profiles between IFN treated and untreated glioblastoma samples were analyzed. Detailed analysis of MS/MS spectra allowed further interpretation of the observed mass shifts and differentiation between post-translational and artifact modifications. Malignant cells typically acquire increased sensitivity to viruses due to the deregulated antiviral mechanisms. Therefore, a viral therapy is considered as one of the promising approaches to treat cancer. However, recent studies have demonstrated that malignant cells can preserve intact antiviral mechanisms, e.g. interferon signaling, and develop resistance to virus infection in response to interferon treatment. Post translational modifications, e.g. tyrosine phosphorylation, are the interferon signaling drivers. Thus, comprehensive characterization of modifications is crucially important, yet, most challenging problem in cancer proteomics. Here, we report on the application of the recently introduced ultra-tolerant search strategy for profiling peptide modifications in the human glioblastoma cell lines demonstrating strong response to the type I interferon (IFNα-2b) treatment. The specific aim of the study was identification of statistically significant changes in peptide mass shift profiles between IFN treated and untreated glioblastoma samples, as well as determination of whether these shifts represent the biologically relevant modification. Copyright © 2018 Elsevier B.V. All rights reserved.

A comparative proteomic strategy for subcellular proteome research: ICAT approach coupled with bioinformatics prediction to ascertain rat liver mitochondrial proteins and indication of mitochondrial localization for catalase.

PubMed

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 proteins and even multilocation proteins. Using such a strategy, many novel proteins, known proteins without subcellular location annotation, and even known proteins that have been annotated as other locations have been strongly indicated for their mitochondrial location.

Spectra-first feature analysis in clinical proteomics - A case study in renal cancer.

PubMed

Goh, Wilson Wen Bin; Wong, Limsoon

2016-10-01

In proteomics, useful signal may be unobserved or lost due to the lack of confident peptide-spectral matches. Selection of differential spectra, followed by associative peptide/protein mapping may be a complementary strategy for improving sensitivity and comprehensiveness of analysis (spectra-first paradigm). This approach is complementary to the standard approach where functional analysis is performed only on the finalized protein list assembled from identified peptides from the spectra (protein-first paradigm). Based on a case study of renal cancer, we introduce a simple spectra-binning approach, MZ-bin. We demonstrate that differential spectra feature selection using MZ-bin is class-discriminative and can trace relevant proteins via spectra associative mapping. Moreover, proteins identified in this manner are more biologically coherent than those selected directly from the finalized protein list. Analysis of constituent peptides per protein reveals high expression inconsistency, suggesting that the measured protein expressions are in fact, poor approximations of true protein levels. Moreover, analysis at the level of constituent peptides may provide higher resolution insight into the underlying biology: Via MZ-bin, we identified for the first time differential splice forms for the known renal cancer marker MAPT. We conclude that the spectra-first analysis paradigm is a complementary strategy to the traditional protein-first paradigm and can provide deeper level insight.

Proteome dynamics of cold-acclimating Rhododendron species contrasting in their freezing tolerance and thermonasty response

USDA-ARS?s Scientific Manuscript database

In the present study we used 2D-DIGE technique to document the Rhododendron proteome during the seasonal development of cold hardiness. We selected two genotypes with different cold hardiness levels. This enabled us to perform comparative analysis of their proteome profiles and screen differentially...

Identification of Major Outer Surface Proteins of Streptococcus agalactiae

PubMed Central

Hughes, Martin J. G.; Moore, Joanne C.; Lane, Jonathan D.; Wilson, Rebecca; Pribul, Philippa K.; Younes, Zabin N.; Dobson, Richard J.; Everest, Paul; Reason, Andrew J.; Redfern, Joanne M.; Greer, Fiona M.; Paxton, Thanai; Panico, Maria; Morris, Howard R.; Feldman, Robert G.; Santangelo, Joseph D.

2002-01-01

To identify the major outer surface proteins of Streptococcus agalactiae (group B streptococcus), a proteomic analysis was undertaken. An extract of the outer surface proteins was separated by two-dimensional electrophoresis. The visualized spots were identified through a combination of peptide sequencing and reverse genetic methodologies. Of the 30 major spots identified as S. agalactiae specific, 27 have been identified. Six of these proteins, previously unidentified in S. agalactiae, were sequenced and cloned. These were ornithine carbamoyltransferase, phosphoglycerate kinase, nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, purine nucleoside phosphorylase, enolase, and glucose-6-phosphate isomerase. Using a gram-positive expression system, we have overexpressed two of these proteins in an in vitro system. These recombinant, purified proteins were used to raise antisera. The identification of these proteins as residing on the outer surface was confirmed by the ability of the antisera to react against whole, live bacteria. Further, in a neonatal-animal model system, we demonstrate that some of these sera are protective against lethal doses of bacteria. These studies demonstrate the successful application of proteomics as a technique for identifying vaccine candidates. PMID:11854208

MASH Suite Pro: A Comprehensive Software Tool for Top-Down Proteomics*

PubMed Central

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Laurence; Yurkovich, James T.; Lloyd, Colton J.

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

Global proteomics profiling improves drug sensitivity prediction: results from a multi-omics, pan-cancer modeling approach.

PubMed

Ali, Mehreen; Khan, Suleiman A; Wennerberg, Krister; Aittokallio, Tero

2018-04-15

Proteomics profiling is increasingly being used for molecular stratification of cancer patients and cell-line panels. However, systematic assessment of the predictive power of large-scale proteomic technologies across various drug classes and cancer types is currently lacking. To that end, we carried out the first pan-cancer, multi-omics comparative analysis of the relative performance of two proteomic technologies, targeted reverse phase protein array (RPPA) and global mass spectrometry (MS), in terms of their accuracy for predicting the sensitivity of cancer cells to both cytotoxic chemotherapeutics and molecularly targeted anticancer compounds. Our results in two cell-line panels demonstrate how MS profiling improves drug response predictions beyond that of the RPPA or the other omics profiles when used alone. However, frequent missing MS data values complicate its use in predictive modeling and required additional filtering, such as focusing on completely measured or known oncoproteins, to obtain maximal predictive performance. Rather strikingly, the two proteomics profiles provided complementary predictive signal both for the cytotoxic and targeted compounds. Further, information about the cellular-abundance of primary target proteins was found critical for predicting the response of targeted compounds, although the non-target features also contributed significantly to the predictive power. The clinical relevance of the selected protein markers was confirmed in cancer patient data. These results provide novel insights into the relative performance and optimal use of the widely applied proteomic technologies, MS and RPPA, which should prove useful in translational applications, such as defining the best combination of omics technologies and marker panels for understanding and predicting drug sensitivities in cancer patients. Processed datasets, R as well as Matlab implementations of the methods are available at https://github.com/mehr-een/bemkl-rbps. mehreen.ali@helsinki.fi or tero.aittokallio@fimm.fi. Supplementary data are available at Bioinformatics online.

Applicability of a high-throughput shotgun plasma protein screening approach in understanding maternal biological pathways relevant to infant birth weight outcome.

PubMed

Kumarathasan, P; Vincent, R; Das, D; Mohottalage, S; Blais, E; Blank, K; Karthikeyan, S; Vuong, N Q; Arbuckle, T E; Fraser, W D

2014-04-04

There are reports linking maternal nutritional status, smoking and environmental chemical exposures to adverse pregnancy outcomes. However, biological bases for association between some of these factors and birth outcomes are yet to be established. The objective of this preliminary work is to test the capability of a new high-throughput shotgun plasma proteomic screening in identifying maternal changes relevant to pregnancy outcome. A subset of third trimester plasma samples (N=12) associated with normal and low-birth weight infants were fractionated, tryptic-digested and analyzed for global proteomic changes using a MALDI-TOF-TOF-MS methodology. Mass spectral data were mined for candidate biomarkers using bioinformatic and statistical tools. Maternal plasma profiles of cytokines (e.g. IL8, TNF-α), chemokines (e.g. MCP-1) and cardiovascular endpoints (e.g. ET-1, MMP-9) were analyzed by a targeted approach using multiplex protein array and HPLC-Fluorescence methods. Target and global plasma proteomic markers were used to identify protein interaction networks and maternal biological pathways relevant to low infant birth weight. Our results exhibited the potential to discriminate specific maternal physiologies relevant to risk of adverse birth outcomes. This proteomic approach can be valuable in understanding the impacts of maternal factors such as environmental contaminant exposures and nutrition on birth outcomes in future work. We demonstrate here the fitness of mass spectrometry-based shot-gun proteomics for surveillance of biological changes in mothers, and for adverse pathway analysis in combination with target biomarker information. This approach has potential for enabling early detection of mothers at risk for low infant birth weight and preterm birth, and thus early intervention for mitigation and prevention of adverse pregnancy outcomes. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes? Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Time-resolved Global and Chromatin Proteomics during Herpes Simplex Virus Type 1 (HSV-1) Infection*

PubMed Central

Kulej, Katarzyna; Avgousti, Daphne C.; Sidoli, Simone; Herrmann, Christin; Della Fera, Ashley N.; Kim, Eui Tae; Garcia, Benjamin A.; Weitzman, Matthew D.

2017-01-01

Herpes simplex virus (HSV-1) lytic infection results in global changes to the host cell proteome and the proteins associated with host chromatin. We present a system level characterization of proteome dynamics during infection by performing a multi-dimensional analysis during HSV-1 lytic infection of human foreskin fibroblast (HFF) cells. Our study includes identification and quantification of the host and viral proteomes, phosphoproteomes, chromatin bound proteomes and post-translational modifications (PTMs) on cellular histones during infection. We analyzed proteomes across six time points of virus infection (0, 3, 6, 9, 12 and 15 h post-infection) and clustered trends in abundance using fuzzy c-means. Globally, we accurately quantified more than 4000 proteins, 200 differently modified histone peptides and 9000 phosphorylation sites on cellular proteins. In addition, we identified 67 viral proteins and quantified 571 phosphorylation events (465 with high confidence site localization) on viral proteins, which is currently the most comprehensive map of HSV-1 phosphoproteome. We investigated chromatin bound proteins by proteomic analysis of the high-salt chromatin fraction and identified 510 proteins that were significantly different in abundance during infection. We found 53 histone marks significantly regulated during virus infection, including a steady increase of histone H3 acetylation (H3K9ac and H3K14ac). Our data provide a resource of unprecedented depth for human and viral proteome dynamics during infection. Collectively, our results indicate that the proteome composition of the chromatin of HFF cells is highly affected during HSV-1 infection, and that phosphorylation events are abundant on viral proteins. We propose that our epi-proteomics approach will prove to be important in the characterization of other model infectious systems that involve changes to chromatin composition. PMID:28179408

Quantitative Proteomics Reveals Temporal Proteomic Changes in Signaling Pathways during BV2 Mouse Microglial Cell Activation.

PubMed

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 discovery and label-free PRM analysis have been deposited to the ProteomeXchange Consortium with identifiers and , respectively.

Comprehensive Proteomic Analysis of Human Milk-derived Extracellular Vesicles Unveils a Novel Functional Proteome Distinct from Other Milk Components*

PubMed Central

van Herwijnen, Martijn J.C.; Zonneveld, Marijke I.; Goerdayal, Soenita; Nolte – 't Hoen, Esther N.M.; Garssen, Johan; Stahl, Bernd; Maarten Altelaar, A.F.; Redegeld, Frank A.; Wauben, Marca H.M.

2016-01-01

Breast milk contains several macromolecular components with distinctive functions, whereby milk fat globules and casein micelles mainly provide nutrition to the newborn, and whey contains molecules that can stimulate the newborn's developing immune system and gastrointestinal tract. Although extracellular vesicles (EV) have been identified in breast milk, their physiological function and composition has not been addressed in detail. EV are submicron sized vehicles released by cells for intercellular communication via selectively incorporated lipids, nucleic acids, and proteins. Because of the difficulty in separating EV from other milk components, an in-depth analysis of the proteome of human milk-derived EV is lacking. In this study, an extensive LC-MS/MS proteomic analysis was performed of EV that had been purified from breast milk of seven individual donors using a recently established, optimized density-gradient-based EV isolation protocol. A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5, and Flotillin-1, with a remarkable overlap between the different donors. Interestingly, 198 of the identified proteins are not present in the human EV database Vesiclepedia, indicating that milk-derived EV harbor proteins not yet identified in EV of different origin. Similarly, the proteome of milk-derived EV was compared with that of other milk components. For this, data from 38 published milk proteomic studies were combined in order to construct the total milk proteome, which consists of 2698 unique proteins. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in human milk to date. Interestingly, these novel proteins include proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, suggesting that milk-derived EVs could support the newborn's developing gastrointestinal tract and immune system. Overall, this study provides an expansion of the whole milk proteome and illustrates that milk-derived EV are macromolecular components with a unique functional proteome. PMID:27601599

Evaluation of the Nicotinic Acetylcholine Receptor-Associated Proteome at Baseline and Following Nicotine Exposure in Human and Mouse Cortex.

PubMed

McClure-Begley, Tristan D; Esterlis, Irina; Stone, Kathryn L; Lam, TuKiet T; Grady, Sharon R; Colangelo, Christopher M; Lindstrom, Jon M; Marks, Michael J; Picciotto, Marina R

2016-01-01

Nicotinic acetylcholine receptors (nAChRs) support the initiation and maintenance of smoking, but the long-term changes occurring in the protein complex as a result of smoking and the nicotine in tobacco are not known. Human studies and animal models have also demonstrated that increasing cholinergic tone increases behaviors related to depression, suggesting that the nAChR-associated proteome could be altered in individuals with mood disorders. We therefore immunopurified nAChRs and associated proteins for quantitative proteomic assessment of changes in protein-protein interactions of high-affinity nAChRs containing the β2 subunit (β2*-nAChRs) from either cortex of mice treated with saline or nicotine, or postmortem human temporal cortex tissue from tobacco-exposed and nonexposed individuals, with a further comparison of diagnosed mood disorder to control subjects. We observed significant effects of nicotine exposure on the β2*-nAChR-associated proteome in human and mouse cortex, particularly in the abundance of the nAChR subunits themselves, as well as putative interacting proteins that make up core components of neuronal excitability (Na/K ATPase subunits), presynaptic neurotransmitter release (syntaxins, SNAP25, synaptotagmin), and a member of a known nAChR protein chaperone family (14-3-3ζ). These findings identify candidate-signaling proteins that could mediate changes in cholinergic signaling via nicotine or tobacco use. Further analysis of identified proteins will determine whether these interactions are essential for primary function of nAChRs at presynaptic terminals. The identification of differences in the nAChR-associated proteome and downstream signaling in subjects with various mood disorders may also identify novel etiological mechanisms and reveal new treatment targets.

Proteomes and Ubiquitylomes Analysis Reveals the Involvement of Ubiquitination in Protein Degradation in Petunias1

PubMed Central

Liu, Juanxu; Wei, Qian; Wang, Rongmin; Yang, Weiyuan; Ma, Yueyue; Chen, Guoju

2017-01-01

Petal senescence is a complex programmed process. It has been demonstrated previously that treatment with ethylene, a plant hormone involved in senescence, can extensively alter transcriptome and proteome profiles in plants. However, little is known regarding the impact of ethylene on posttranslational modification (PTM) or the association between PTM and the proteome. Protein degradation is one of the hallmarks of senescence, and ubiquitination, a major PTM in eukaryotes, plays important roles in protein degradation. In this study, we first obtained reference petunia (Petunia hybrida) transcriptome data via RNA sequencing. Next, we quantitatively investigated the petunia proteome and ubiquitylome and the association between them in petunia corollas following ethylene treatment. In total, 51,799 unigenes, 3,606 proteins, and 2,270 ubiquitination sites were quantified 16 h after ethylene treatment. Treatment with ethylene resulted in 14,448 down-regulated and 6,303 up-regulated unigenes (absolute log2 fold change > 1 and false discovery rate < 0.001), 284 down-regulated and 233 up-regulated proteins, and 320 up-regulated and 127 down-regulated ubiquitination sites using a 1.5-fold threshold (P < 0.05), indicating that global ubiquitination levels increase during ethylene-mediated corolla senescence in petunia. Several putative ubiquitin ligases were up-regulated at the protein and transcription levels. Our results showed that the global proteome and ubiquitylome were negatively correlated and that ubiquitination could be involved in the degradation of proteins during ethylene-mediated corolla senescence in petunia. Ethylene regulates hormone signaling transduction pathways at both the protein and ubiquitination levels in petunia corollas. In addition, our results revealed that ethylene increases the ubiquitination levels of proteins involved in endoplasmic reticulum-associated degradation. PMID:27810942

Proteomic approaches and their application to plant gravitropism.

PubMed

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.

Ultraviolet photodissociation enhances top-down mass spectrometry as demonstrated on green fluorescent protein variants.

PubMed

Dang, Xibei; Young, Nicolas L

2014-05-01

Ultraviolet photodissociation (UVPD) is a compelling fragmentation technique with great potential to enhance proteomics generally and top-down MS specifically. In this issue, Cannon et al. (Proteomics 2014, 14, XXXX-XXXX) use UVPD to perform top-down MS on several sequence variants of green fluorescent protein and compare the results to CID, higher energy collision induced dissociation, and electron transfer dissociation. As compared to the other techniques UVPD produces a wider variety of fragment ion types that are relatively evenly distributed across the protein sequences. Overall, their results demonstrate enhanced sequence coverage and higher confidence in sequence assignment via UVPD MS. Based on these and other recent results UVPD is certain to become an increasingly widespread and valuable tool for top-down proteomics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic Profiling of Cranial (Superior) Cervical Ganglia Reveals Beta-Amyloid and Ubiquitin Proteasome System Perturbations in an Equine Multiple System Neuropathy.

PubMed

McGorum, Bruce C; Pirie, R Scott; Eaton, Samantha L; Keen, John A; Cumyn, Elizabeth M; Arnott, Danielle M; Chen, Wenzhang; Lamont, Douglas J; Graham, Laura C; Llavero Hurtado, Maica; Pemberton, Alan; Wishart, Thomas M

2015-11-01

Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell-to-cell signaling and interaction (including epinephrine, dopamine, and adrenergic signaling and receptor function), and small molecule biochemistry. Interestingly, while the biofunctions identified in this study may represent pathways underpinning EGS-induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to small animal models with altered neuronal vulnerability, and human neurological conditions. Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Application of Large-Scale Aptamer-Based Proteomic Profiling to Planned Myocardial Infarctions.

PubMed

Jacob, Jaison; Ngo, Debby; Finkel, Nancy; Pitts, Rebecca; Gleim, Scott; Benson, Mark D; Keyes, Michelle J; Farrell, Laurie A; Morgan, Thomas; Jennings, Lori L; Gerszten, Robert E

2018-03-20

Emerging proteomic technologies using novel affinity-based reagents allow for efficient multiplexing with high-sample throughput. To identify early biomarkers of myocardial injury, we recently applied an aptamer-based proteomic profiling platform that measures 1129 proteins to samples from patients undergoing septal alcohol ablation for hypertrophic cardiomyopathy, a human model of planned myocardial injury. Here, we examined the scalability of this approach using a markedly expanded platform to study a far broader range of human proteins in the context of myocardial injury. We applied a highly multiplexed, expanded proteomic technique that uses single-stranded DNA aptamers to assay 4783 human proteins (4137 distinct human gene targets) to derivation and validation cohorts of planned myocardial injury, individuals with spontaneous myocardial infarction, and at-risk controls. We found 376 target proteins that significantly changed in the blood after planned myocardial injury in a derivation cohort (n=20; P <1.05E-05, 1-way repeated measures analysis of variance, Bonferroni threshold). Two hundred forty-seven of these proteins were validated in an independent planned myocardial injury cohort (n=15; P <1.33E-04, 1-way repeated measures analysis of variance); >90% were directionally consistent and reached nominal significance in the validation cohort. Among the validated proteins that were increased within 1 hour after planned myocardial injury, 29 were also elevated in patients with spontaneous myocardial infarction (n=63; P <6.17E-04). Many of the novel markers identified in our study are intracellular proteins not previously identified in the peripheral circulation or have functional roles relevant to myocardial injury. For example, the cardiac LIM protein, cysteine- and glycine-rich protein 3, is thought to mediate cardiac mechanotransduction and stress responses, whereas the mitochondrial ATP synthase F 0 subunit component is a vasoactive peptide on its release from cells. Last, we performed aptamer-affinity enrichment coupled with mass spectrometry to technically verify aptamer specificity for a subset of the new biomarkers. Our results demonstrate the feasibility of large-scale aptamer multiplexing at a level that has not previously been reported and with sample throughput that greatly exceeds other existing proteomic methods. The expanded aptamer-based proteomic platform provides a unique opportunity for biomarker and pathway discovery after myocardial injury. © 2017 American Heart Association, Inc.

Announcing the Launch of CPTAC’s Proteogenomics DREAM Challenge | Office of Cancer Clinical Proteomics Research

Cancer.gov

This week, we are excited to announce the launch of the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) Proteogenomics Computational DREAM Challenge.  The aim of this Challenge is to encourage the generation of computational methods for extracting information from the cancer proteome and for linking those data to genomic and transcriptomic information.  The specific goals are to predict proteomic and phosphoproteomic data from other multiple data types including transcriptomics and genetics.

GENOMIC AND PROTEOMIC ANALYSIS OF SURROGATE TISSUES FOR ASSESSING TOXIC EXPOSURES AND DISEASE STATES

EPA Science Inventory

Genomic and Proteomic Analysis of Surrogate Tissues for Assessing Toxic Exposures and Disease States
David J. Dix and John C. Rockett
Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, USEPA, ...

DAnTE: a statistical tool for quantitative analysis of –omics data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Polpitiya, Ashoka D.; Qian, Weijun; Jaitly, Navdeep

2008-05-03

DAnTE (Data Analysis Tool Extension) is a statistical tool designed to address challenges unique to quantitative bottom-up, shotgun proteomics data. This tool has also been demonstrated for microarray data and can easily be extended to other high-throughput data types. DAnTE features selected normalization methods, missing value imputation algorithms, peptide to protein rollup methods, an extensive array of plotting functions, and a comprehensive ANOVA scheme that can handle unbalanced data and random effects. The Graphical User Interface (GUI) is designed to be very intuitive and user friendly.

Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

PubMed Central

Whigham, Arlene; Clarke, Rosemary; Brenes-Murillo, Alejandro J; Estes, Brett; Madhessian, Diana; Lundberg, Emma; Wadsworth, Patricia

2017-01-01

The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al., 2014, 2015). Here, we show that by using specific intracellular immunolabelling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabelled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, termed ‘early risers’. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide the first a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase. PMID:29052541

A DIGE proteomic analysis for high-intensity exercise-trained rat skeletal muscle.

PubMed

Yamaguchi, Wataru; Fujimoto, Eri; Higuchi, Mitsuru; Tabata, Izumi

2010-09-01

Exercise training induces various adaptations in skeletal muscles. However, the mechanisms remain unclear. In this study, we conducted 2D-DIGE proteomic analysis, which has not yet been used for elucidating adaptations of skeletal muscle after high-intensity exercise training (HIT). For 5 days, rats performed HIT, which consisted of 14 20-s swimming exercise bouts carrying a weight (14% of the body weight), and 10-s pause between bouts. The 2D-DIGE analysis was conducted on epitrochlearis muscles excised 18 h after the final training exercise. Proteomic profiling revealed that out of 800 detected and matched spots, 13 proteins exhibited changed expression by HIT compared with sedentary rats. All proteins were identified by MALDI-TOF/MS. Furthermore, using western immunoblot analyses, significantly changed expressions of NDUFS1 and parvalbumin (PV) were validated in relation to HIT. In conclusion, the proteomic 2D-DIGE analysis following HIT-identified expressions of NDUFS1 and PV, previously unknown to have functions related to exercise-training adaptations.

Progress in Top-Down Proteomics and the Analysis of Proteoforms

PubMed Central

Toby, Timothy K.; Fornelli, Luca; Kelleher, Neil L.

2017-01-01

From a molecular perspective, enactors of function in biology are intact proteins that can be variably modified at the genetic, transcriptional, or post-translational level. Over the past 30 years, mass spectrometry (MS) has become a powerful method for the analysis of proteomes. Prevailing bottom-up proteomics operates at the level of the peptide, leading to issues with protein inference, connectivity, and incomplete sequence/modification information. Top-down proteomics (TDP), alternatively, applies MS at the proteoform level to analyze intact proteins with diverse sources of intramolecular complexity preserved during analysis. Fortunately, advances in prefractionation workflows, MS instrumentation, and dissociation methods for whole-protein ions have helped TDP emerge as an accessible and potentially disruptive modality with increasingly translational value. In this review, we discuss technical and conceptual advances in TDP, along with the growing power of proteoform-resolved measurements in clinical and translational research. PMID:27306313

Sex-Specific Biology of the Human Malaria Parasite Revealed from the Proteomes of Mature Male and Female Gametocytes.

PubMed

Miao, Jun; Chen, Zhao; Wang, Zenglei; Shrestha, Sony; Li, Xiaolian; Li, Runze; Cui, Liwang

2017-04-01

The gametocytes of the malaria parasites are obligate for perpetuating the parasite's life cycle through mosquitoes, but the sex-specific biology of gametocytes is poorly understood. We generated a transgenic line in the human malaria parasite Plasmodium falciparum , which allowed us to accurately separate male and female gametocytes by flow cytometry. In-depth analysis of the proteomes by liquid chromatography-tandem mass spectrometry identified 1244 and 1387 proteins in mature male and female gametocytes, respectively. GFP-tagging of nine selected proteins confirmed their sex-partitions to be agreeable with the results from the proteomic analysis. The sex-specific proteomes showed significant differences that are consistent with the divergent functions of the two sexes. Although the male-specific proteome (119 proteins) is enriched in proteins associated with the flagella and genome replication, the female-specific proteome (262 proteins) is more abundant in proteins involved in metabolism, translation and organellar functions. Compared with the Plasmodium berghei sex-specific proteomes, this study revealed both extensive conservation and considerable divergence between these two species, which reflect the disparities between the two species in proteins involved in cytoskeleton, lipid metabolism and protein degradation. Comparison with three sex-specific proteomes allowed us to obtain high-confidence lists of 73 and 89 core male- and female-specific/biased proteins conserved in Plasmodium The identification of sex-specific/biased proteomes in Plasmodium lays a solid foundation for understanding the molecular mechanisms underlying the unique sex-specific biology in this early-branching eukaryote. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Sex-Specific Biology of the Human Malaria Parasite Revealed from the Proteomes of Mature Male and Female Gametocytes *

PubMed Central

Miao, Jun; Chen, Zhao; Wang, Zenglei; Shrestha, Sony; Li, Xiaolian; Li, Runze; Cui, Liwang

2017-01-01

The gametocytes of the malaria parasites are obligate for perpetuating the parasite's life cycle through mosquitoes, but the sex-specific biology of gametocytes is poorly understood. We generated a transgenic line in the human malaria parasite Plasmodium falciparum, which allowed us to accurately separate male and female gametocytes by flow cytometry. In-depth analysis of the proteomes by liquid chromatography-tandem mass spectrometry identified 1244 and 1387 proteins in mature male and female gametocytes, respectively. GFP-tagging of nine selected proteins confirmed their sex-partitions to be agreeable with the results from the proteomic analysis. The sex-specific proteomes showed significant differences that are consistent with the divergent functions of the two sexes. Although the male-specific proteome (119 proteins) is enriched in proteins associated with the flagella and genome replication, the female-specific proteome (262 proteins) is more abundant in proteins involved in metabolism, translation and organellar functions. Compared with the Plasmodium berghei sex-specific proteomes, this study revealed both extensive conservation and considerable divergence between these two species, which reflect the disparities between the two species in proteins involved in cytoskeleton, lipid metabolism and protein degradation. Comparison with three sex-specific proteomes allowed us to obtain high-confidence lists of 73 and 89 core male- and female-specific/biased proteins conserved in Plasmodium. The identification of sex-specific/biased proteomes in Plasmodium lays a solid foundation for understanding the molecular mechanisms underlying the unique sex-specific biology in this early-branching eukaryote. PMID:28126901

Proteomic analysis of oil bodies in mature Jatropha curcas seeds with different lipid content.

PubMed

Liu, Hui; Wang, Cuiping; Chen, Fan; Shen, Shihua

2015-01-15

To reveal the difference among three mature Jatropha curcas seeds (JcVH, variant with high lipid content; JcW, wild type and JcVL, variant with low lipid content) with different lipid content, comparative proteomics was employed to profile the changes of oil body (OB) associated protein species by using gels-based proteomic technique. Eighty-three protein species were successfully identified through LTQ-ES-MS/MS from mature JcW seeds purified OBs. Two-dimensional electrophoresis analysis of J. curcas OB associated protein species revealed they had essential interactions with other organelles and demonstrated that oleosin and caleosin were the most abundant OB structural protein species. Twenty-eight OB associated protein species showed significant difference among JcVH, JcW and JcVL according to statistical analysis. Complementary transient expression analysis revealed that calcium ion binding protein (CalBP) and glycine-rich RNA binding protein (GRP) were well targeted in OBs apart from the oleosins. This study demonstrated that ratio of lipid content to caleosins abundance was involved in the regulation of OB size, and the mutant induced by ethylmethylsulfone treatment might be related to the caleosin like protein species. These findings are important for biotechnological improvement with the aim to alter the lipid content in J. curcas seeds. The economic value of Jatropha curcas largely depends on the lipid content in seeds which are mainly stored in the special organelle called oil bodies (OBs). In consideration of the biological importance and applications of J. curcas OB in seeds, it is necessary to further explore the components and functions of J. curcas OBs. Although a previous study concerning the J. curcas OB proteome revealed oleosins were the major OB protein component and additional protein species were similar to those in other oil seed plants, these identified OB associated protein species were corresponding to the protein bands instead of protein spots in the electrophoresis gels. Furthermore, the interaction of OB associated protein species and their contribution to OB formation and stabilization are still blank. In this study, with the overall object of profiling OB protein species from mature J. curcas seeds with different lipid content, we provided a setting of comparative OB proteomics with biochemical data and transient expression to explore the core of OB associated protein species involved in the regulation of OB size and lipid accumulation. The results were important for biotechnological improvement with the aim to a global modification of lipid storage in J. curcas seeds. Meanwhile, this study gave insight into possible associations between OBs and other organelles in mature J. curcas seeds. It may represent new aspects of the biological functions of the OBs during the oil mobilization. Combined the technique of transient transformation, a newly reported protein species, glycine-rich RNA binding protein (GRP) was successfully targeted in OBs. Therefore, further molecular analysis of these protein species is warranted to verify this association and what role they have in OBs. Copyright © 2014 Elsevier B.V. All rights reserved.

Cell death proteomics database: consolidating proteomics data on cell death.

PubMed

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.

MitoMiner: a data warehouse for mitochondrial proteomics data

PubMed Central

Smith, Anthony C.; Blackshaw, James A.; Robinson, Alan J.

2012-01-01

MitoMiner (http://mitominer.mrc-mbu.cam.ac.uk/) is a data warehouse for the storage and analysis of mitochondrial proteomics data gathered from publications of mass spectrometry and green fluorescent protein tagging studies. In MitoMiner, these data are integrated with data from UniProt, Gene Ontology, Online Mendelian Inheritance in Man, HomoloGene, Kyoto Encyclopaedia of Genes and Genomes and PubMed. The latest release of MitoMiner stores proteomics data sets from 46 studies covering 11 different species from eumetazoa, viridiplantae, fungi and protista. MitoMiner is implemented by using the open source InterMine data warehouse system, which provides a user interface allowing users to upload data for analysis, personal accounts to store queries and results and enables queries of any data in the data model. MitoMiner also provides lists of proteins for use in analyses, including the new MitoMiner mitochondrial proteome reference sets that specify proteins with substantial experimental evidence for mitochondrial localization. As further mitochondrial proteomics data sets from normal and diseased tissue are published, MitoMiner can be used to characterize the variability of the mitochondrial proteome between tissues and investigate how changes in the proteome may contribute to mitochondrial dysfunction and mitochondrial-associated diseases such as cancer, neurodegenerative diseases, obesity, diabetes, heart failure and the ageing process. PMID:22121219

The Challenge of Human Spermatozoa Proteome: A Systematic Review.

PubMed

Gilany, Kambiz; Minai-Tehrani, Arash; Amini, Mehdi; Agharezaee, Niloofar; Arjmand, Babak

2017-01-01

Currently, there are 20,197 human protein-coding genes in the most expertly curated database (UniProtKB/Swiss-Pro). Big efforts have been made by the international consortium, the Chromosome-Centric Human Proteome Project (C-HPP) and independent researchers, to map human proteome. In brief, anno 2017 the human proteome was outlined. The male factor contributes to 50% of infertility in couples. However, there are limited human spermatozoa proteomic studies. Firstly, the development of the mapping of the human spermatozoa was analyzed. The human spermatozoa have been used as a model for missing proteins. It has been shown that human spermatozoa are excellent sources for finding missing proteins. Y chromosome proteome mapping is led by Iran. However, it seems that it is extremely challenging to map the human spermatozoa Y chromosome proteins based on current mass spectrometry-based proteomics technology. Post-translation modifications (PTMs) of human spermatozoa proteome are the most unexplored area and currently the exact role of PTMs in male infertility is unknown. Additionally, the clinical human spermatozoa proteomic analysis, anno 2017 was done in this study.

PatternLab for proteomics 4.0: A one-stop shop for analyzing shotgun proteomic data

PubMed Central

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

Computer applications making rapid advances in high throughput microbial proteomics (HTMP).

PubMed

Anandkumar, Balakrishna; Haga, Steve W; Wu, Hui-Fen

2014-02-01

The last few decades have seen the rise of widely-available proteomics tools. From new data acquisition devices, such as MALDI-MS and 2DE to new database searching softwares, these new products have paved the way for high throughput microbial proteomics (HTMP). These tools are enabling researchers to gain new insights into microbial metabolism, and are opening up new areas of study, such as protein-protein interactions (interactomics) discovery. Computer software is a key part of these emerging fields. This current review considers: 1) software tools for identifying the proteome, such as MASCOT or PDQuest, 2) online databases of proteomes, such as SWISS-PROT, Proteome Web, or the Proteomics Facility of the Pathogen Functional Genomics Resource Center, and 3) software tools for applying proteomic data, such as PSI-BLAST or VESPA. These tools allow for research in network biology, protein identification, functional annotation, target identification/validation, protein expression, protein structural analysis, metabolic pathway engineering and drug discovery.

The effect of electroacupuncture on proteomic changes in the motor cortex of 6-OHDA Parkinsonian rats.

PubMed

Li, Min; Li, Lijuan; Wang, Ke; Su, Wenting; Jia, Jun; Wang, Xiaomin

2017-10-15

Electroacupuncture (EA) has been reported to alleviate motor deficits in Parkinson's disease (PD) patients, and PD animal models. However, the mechanisms by which EA improves motor function have not been investigated. We have employed a 6-hydroxydopamine (6-OHDA) unilateral injection induced PD model to investigate whether EA alters protein expression in the motor cortex. We found that 4weeks of EA treatment significantly improved spontaneous floor plane locomotion and rotarod performance. High-throughput proteomic analysis in the motor cortex was employed. The expression of 54 proteins were altered in the unlesioned motor cortex, and 102 protein expressions were altered in the lesioned motor cortex of 6-OHDA rats compared to sham rats. Compared to non-treatment PD control, EA treatment reversed 6 proteins in unlesioned and 19 proteins in lesioned motor cortex. The present study demonstrated that PD induces proteomic changes in the motor cortex, some of which are rescued by EA treatment. These targeted proteins were mainly involved in increasing autophagy, mRNA processing and ATP binding and maintaining the balance of neurotransmitters. Copyright © 2017 Elsevier B.V. All rights reserved.

Study of cellular oncometabolism via multidimensional protein identification technology.

PubMed

Aukim-Hastie, Claire; Garbis, Spiros D

2014-01-01

Cellular proteomics is becoming a widespread clinical application, matching the definition of bench-to-bedside translation. Among various fields of investigation, this approach can be applied to the study of the metabolic alterations that accompany oncogenesis and tumor progression, which are globally referred to as oncometabolism. Here, we describe a multidimensional protein identification technology (MuDPIT)-based strategy that can be employed to study the cellular proteome of malignant cells and tissues. This method has previously been shown to be compatible with the reproducible, in-depth analysis of up to a thousand proteins in clinical samples. The possibility to employ this technique to study clinical specimens demonstrates its robustness. MuDPIT is advantageous as compared to other approaches because it is direct, highly sensitive, and reproducible, it provides high resolution with ultra-high mass accuracy, it allows for relative quantifications, and it is compatible with multiplexing (thus limiting costs).This method enables the direct assessment of the proteomic profile of neoplastic cells and tissues and could be employed in the near future as a high-throughput, rapid, quantitative, and cost-effective screening platform for clinical samples. © 2014 Elsevier Inc. All rights reserved.