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.

State-of-the-art nanoplatform-integrated MALDI-MS impacting resolutions in urinary proteomics.

PubMed

Gopal, Judy; Muthu, Manikandan; Chun, Se-Chul; Wu, Hui-Fen

2015-06-01

Urine proteomics has become a subject of interest, since it has led to a number of breakthroughs in disease diagnostics. Urine contains information not only from the kidney and the urinary tract but also from other organs, thus urinary proteome analysis allows for identification of biomarkers for both urogenital and systemic diseases. The following review gives a brief overview of the analytical techniques that have been in practice for urinary proteomics. MALDI-MS technique and its current application status in this area of clinical research have been discussed. The review comments on the challenges facing the conventional MALDI-MS technique and the upgradation of this technique with the introduction of nanotechnology. This review projects nano-based techniques such as nano-MALDI-MS, surface-assisted laser desorption/ionization, and nanostructure-initiator MS as the platforms that have the potential in trafficking MALDI-MS from the lab to the bedside. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mass spectrometry-based proteomics: basic principles and emerging technologies and directions.

PubMed

Van Riper, Susan K; de Jong, Ebbing P; Carlis, John V; Griffin, Timothy J

2013-01-01

As the main catalytic and structural molecules within living systems, proteins are the most likely biomolecules to be affected by radiation exposure. Proteomics, the comprehensive characterization of proteins within complex biological samples, is therefore a research approach ideally suited to assess the effects of radiation exposure on cells and tissues. For comprehensive characterization of proteomes, an analytical platform capable of quantifying protein abundance, identifying post-translation modifications and revealing members of protein complexes on a system-wide level is necessary. Mass spectrometry (MS), coupled with technologies for sample fractionation and automated data analysis, provides such a versatile and powerful platform. In this chapter we offer a view on the current state of MS-proteomics, and focus on emerging technologies within three areas: (1) New instrumental methods; (2) New computational methods for peptide identification; and (3) Label-free quantification. These emerging technologies should be valuable for researchers seeking to better understand biological effects of radiation on living systems.

Highlights of the Biology and Disease-driven Human Proteome Project, 2015-2016.

PubMed

Van Eyk, Jennifer E; Corrales, Fernando J; Aebersold, Ruedi; Cerciello, Ferdinando; Deutsch, Eric W; Roncada, Paola; Sanchez, Jean-Charles; Yamamoto, Tadashi; Yang, Pengyuan; Zhang, Hui; Omenn, Gilbert S

2016-11-04

The Biology and Disease-driven Human Proteome Project (B/D-HPP) is aimed at supporting and enhancing the broad use of state-of-the-art proteomic methods to characterize and quantify proteins for in-depth understanding of the molecular mechanisms of biological processes and human disease. Based on a foundation of the pre-existing HUPO initiatives begun in 2002, the B/D-HPP is designed to provide standardized methods and resources for mass spectrometry and specific protein affinity reagents and facilitate accessibility of these resources to the broader life sciences research and clinical communities. Currently there are 22 B/D-HPP initiatives and 3 closely related HPP resource pillars. The B/D-HPP groups are working to define sets of protein targets that are highly relevant to each particular field to deliver relevant assays for the measurement of these selected targets and to disseminate and make publicly accessible the information and tools generated. Major developments are the 2016 publications of the Human SRM Atlas and of "popular protein sets" for six organ systems. Here we present the current activities and plans of the BD-HPP initiatives as highlighted in numerous B/D-HPP workshops at the 14th annual HUPO 2015 World Congress of Proteomics in Vancouver, Canada.

Proteomic approach to nanotoxicity.

PubMed

Matysiak, Magdalena; Kapka-Skrzypczak, Lucyna; Brzóska, Kamil; Gutleb, Arno C; Kruszewski, Marcin

2016-03-30

In recent years a large number of engineered nanomaterials (NMs) have been developed with promising technical benefits for consumers and medical appliances. In addition to already known potentially advantageous biological properties (antibiotic, antifungal and antiviral activity) of NMs, many new medical applications of NMs are foreseen, such as drug carriers, contrast agents, radiopharmaceuticals and many others. However, there is increasing concern about potential environmental and health effects due to NMs exposure. An increasing body of evidence suggests that NMs may trigger undesirable hazardous interactions with biological systems with potential to generate harmful effects. In this review we summarized a current state of knowledge on the proteomics approaches to nanotoxicity, including protein corona formation, in vitro and in vivo effects of exposure to NMs on proteome of different classes of organisms, from bacteria and plants to mammals. The effects of NMs on the proteome of environmentally relevant organisms are also described. Despite the benefit that development of nanotechnology may bring to the society, there are still major gaps of knowledge on the influence of nanomaterials on human health and the environment. Thus, it seems necessary to conduct further interdisciplinary research to fill the knowledge gaps in NM toxicity, using more holistic approaches than offered by conventional biological techniques. “OMICS” techniques will certainly help researchers in this field. In this paper we summarized the current stage of knowledge of the effects of nanoparticles on the proteome of different organisms, including those commonly used as an environmentally relevant indicator organisms.

Current state of the art for enhancing urine biomarker discovery

PubMed Central

Harpole, Michael; Davis, Justin; Espina, Virginia

2016-01-01

Urine is a highly desirable biospecimen for biomarker analysis because it can be collected recurrently by non-invasive techniques, in relatively large volumes. Urine contains cellular elements, biochemicals, and proteins derived from glomerular filtration of plasma, renal tubule excretion, and urogenital tract secretions that reflect, at a given time point, an individual's metabolic and pathophysiologic state. High-resolution mass spectrometry, coupled with state of the art fractionation systems are revealing the plethora of diagnostic/prognostic proteomic information existing within urinary exosomes, glycoproteins, and proteins. Affinity capture pre-processing techniques such as combinatorial peptide ligand libraries and biomarker harvesting hydrogel nanoparticles are enabling measurement/identification of previously undetectable urinary proteins. Future challenges in the urinary proteomics field include a) defining either single or multiple, universally applicable data normalization methods for comparing results within and between individual patients/data sets, and b) defining expected urinary protein levels in healthy individuals. PMID:27232439

Clinical proteomics-driven precision medicine for targeted cancer therapy: current overview and future perspectives.

PubMed

Zhou, Li; Wang, Kui; Li, Qifu; Nice, Edouard C; Zhang, Haiyuan; Huang, Canhua

2016-01-01

Cancer is a common disease that is a leading cause of death worldwide. Currently, early detection and novel therapeutic strategies are urgently needed for more effective management of cancer. Importantly, protein profiling using clinical proteomic strategies, with spectacular sensitivity and precision, offer excellent promise for the identification of potential biomarkers that would direct the development of targeted therapeutic anticancer drugs for precision medicine. In particular, clinical sample sources, including tumor tissues and body fluids (blood, feces, urine and saliva), have been widely investigated using modern high-throughput mass spectrometry-based proteomic approaches combined with bioinformatic analysis, to pursue the possibilities of precision medicine for targeted cancer therapy. Discussed in this review are the current advantages and limitations of clinical proteomics, the available strategies of clinical proteomics for the management of precision medicine, as well as the challenges and future perspectives of clinical proteomics-driven precision medicine for targeted cancer therapy.

Environmental proteomics: a paradigm shift in characterizing microbial activities at the molecular level.

PubMed

Keller, Martin; Hettich, Robert

2009-03-01

The increase in sequencing capacity led to a new wave of metagenomic projects, enabling and setting the prerequisite for the application of environmental proteomics technologies. This review describes the current status of environmental proteomics. It describes sample preparation as well as the two major technologies applied within this field: two-dimensional electrophoresis-based environmental proteomics and liquid chromatography-mass spectrometry-based environmental proteomics. It also highlights current publications and describes major scientific findings. The review closes with a discussion of critical improvements in the area of integrating experimental mass spectrometry technologies with bioinformatics as well as improved sample handling.

Current trends in quantitative proteomics - an update.

PubMed

Li, H; Han, J; Pan, J; Liu, T; Parker, C E; Borchers, C H

2017-05-01

Proteins can provide insights into biological processes at the functional level, so they are very promising biomarker candidates. The quantification of proteins in biological samples has been routinely used for the diagnosis of diseases and monitoring the treatment. Although large-scale protein quantification in complex samples is still a challenging task, a great amount of effort has been made to advance the technologies that enable quantitative proteomics. Seven years ago, in 2009, we wrote an article about the current trends in quantitative proteomics. In writing this current paper, we realized that, today, we have an even wider selection of potential tools for quantitative proteomics. These tools include new derivatization reagents, novel sampling formats, new types of analyzers and scanning techniques, and recently developed software to assist in assay development and data analysis. In this review article, we will discuss these innovative methods, and their current and potential applications in proteomics. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Comparative shotgun proteomics using spectral count data and quasi-likelihood modeling.

PubMed

Li, Ming; Gray, William; Zhang, Haixia; Chung, Christine H; Billheimer, Dean; Yarbrough, Wendell G; Liebler, Daniel C; Shyr, Yu; Slebos, Robbert J C

2010-08-06

Shotgun proteomics provides the most powerful analytical platform for global inventory of complex proteomes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and allows a global analysis of protein changes. Nevertheless, sampling of complex proteomes by current shotgun proteomics platforms is incomplete, and this contributes to variability in assessment of peptide and protein inventories by spectral counting approaches. Thus, shotgun proteomics data pose challenges in comparing proteomes from different biological states. We developed an analysis strategy using quasi-likelihood Generalized Linear Modeling (GLM), included in a graphical interface software package (QuasiTel) that reads standard output from protein assemblies created by IDPicker, an HTML-based user interface to query shotgun proteomic data sets. This approach was compared to four other statistical analysis strategies: Student t test, Wilcoxon rank test, Fisher's Exact test, and Poisson-based GLM. We analyzed the performance of these tests to identify differences in protein levels based on spectral counts in a shotgun data set in which equimolar amounts of 48 human proteins were spiked at different levels into whole yeast lysates. Both GLM approaches and the Fisher Exact test performed adequately, each with their unique limitations. We subsequently compared the proteomes of normal tonsil epithelium and HNSCC using this approach and identified 86 proteins with differential spectral counts between normal tonsil epithelium and HNSCC. We selected 18 proteins from this comparison for verification of protein levels between the individual normal and tumor tissues using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS). This analysis confirmed the magnitude and direction of the protein expression differences in all 6 proteins for which reliable data could be obtained. Our analysis demonstrates that shotgun proteomic data sets from different tissue phenotypes are sufficiently rich in quantitative information and that statistically significant differences in proteins spectral counts reflect the underlying biology of the samples.

Comparative Shotgun Proteomics Using Spectral Count Data and Quasi-Likelihood Modeling

PubMed Central

2010-01-01

Shotgun proteomics provides the most powerful analytical platform for global inventory of complex proteomes using liquid chromatography−tandem mass spectrometry (LC−MS/MS) and allows a global analysis of protein changes. Nevertheless, sampling of complex proteomes by current shotgun proteomics platforms is incomplete, and this contributes to variability in assessment of peptide and protein inventories by spectral counting approaches. Thus, shotgun proteomics data pose challenges in comparing proteomes from different biological states. We developed an analysis strategy using quasi-likelihood Generalized Linear Modeling (GLM), included in a graphical interface software package (QuasiTel) that reads standard output from protein assemblies created by IDPicker, an HTML-based user interface to query shotgun proteomic data sets. This approach was compared to four other statistical analysis strategies: Student t test, Wilcoxon rank test, Fisher’s Exact test, and Poisson-based GLM. We analyzed the performance of these tests to identify differences in protein levels based on spectral counts in a shotgun data set in which equimolar amounts of 48 human proteins were spiked at different levels into whole yeast lysates. Both GLM approaches and the Fisher Exact test performed adequately, each with their unique limitations. We subsequently compared the proteomes of normal tonsil epithelium and HNSCC using this approach and identified 86 proteins with differential spectral counts between normal tonsil epithelium and HNSCC. We selected 18 proteins from this comparison for verification of protein levels between the individual normal and tumor tissues using liquid chromatography−multiple reaction monitoring mass spectrometry (LC−MRM-MS). This analysis confirmed the magnitude and direction of the protein expression differences in all 6 proteins for which reliable data could be obtained. Our analysis demonstrates that shotgun proteomic data sets from different tissue phenotypes are sufficiently rich in quantitative information and that statistically significant differences in proteins spectral counts reflect the underlying biology of the samples. PMID:20586475

Proteomic analysis of a model fish species exposed to individual pesticides and a binary mixture--Presentation

EPA Science Inventory

Pesticides are nearly ubiquitous in surface waters of the United States, where they often are found as mixtures. The molecular mechanisms underlying the toxic effects of sub-lethal exposure to pesticides as both individual and mixtures are unclear. The current work aims to ident...

Clinical veterinary proteomics: Techniques and approaches to decipher the animal plasma proteome.

PubMed

Ghodasara, P; Sadowski, P; Satake, N; Kopp, S; Mills, P C

2017-12-01

Over the last two decades, technological advancements in the field of proteomics have advanced our understanding of the complex biological systems of living organisms. Techniques based on mass spectrometry (MS) have emerged as powerful tools to contextualise existing genomic information and to create quantitative protein profiles from plasma, tissues or cell lines of various species. Proteomic approaches have been used increasingly in veterinary science to investigate biological processes responsible for growth, reproduction and pathological events. However, the adoption of proteomic approaches by veterinary investigators lags behind that of researchers in the human medical field. Furthermore, in contrast to human proteomics studies, interpretation of veterinary proteomic data is difficult due to the limited protein databases available for many animal species. This review article examines the current use of advanced proteomics techniques for evaluation of animal health and welfare and covers the current status of clinical veterinary proteomics research, including successful protein identification and data interpretation studies. It includes a description of an emerging tool, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS), available on selected mass spectrometry instruments. This newly developed data acquisition technique combines advantages of discovery and targeted proteomics approaches, and thus has the potential to advance the veterinary proteomics field by enhancing identification and reproducibility of proteomics data. Copyright © 2017 Elsevier Ltd. All rights reserved.

Plant proteomics in India and Nepal: current status and challenges ahead.

PubMed

Deswal, Renu; Gupta, Ravi; Dogra, Vivek; Singh, Raksha; Abat, Jasmeet Kaur; Sarkar, Abhijit; Mishra, Yogesh; Rai, Vandana; Sreenivasulu, Yelam; Amalraj, Ramesh Sundar; Raorane, Manish; Chaudhary, Ram Prasad; Kohli, Ajay; Giri, Ashok Prabhakar; Chakraborty, Niranjan; Zargar, Sajad Majeed; Agrawal, Vishwanath Prasad; Agrawal, Ganesh Kumar; Job, Dominique; Renaut, Jenny; Rakwal, Randeep

2013-10-01

Plant proteomics has made tremendous contributions in understanding the complex processes of plant biology. Here, its current status in India and Nepal is discussed. Gel-based proteomics is predominantly utilized on crops and non-crops to analyze majorly abiotic (49 %) and biotic (18 %) stress, development (11 %) and post-translational modifications (7 %). Rice is the most explored system (36 %) with major focus on abiotic mainly dehydration (36 %) stress. In spite of expensive proteomics setup and scarcity of trained workforce, output in form of publications is encouraging. To boost plant proteomics in India and Nepal, researchers have discussed ground level issues among themselves and with the International Plant Proteomics Organization (INPPO) to act in priority on concerns like food security. Active collaboration may help in translating this knowledge to fruitful applications.

Current advances in esophageal cancer proteomics.

PubMed

Uemura, Norihisa; Kondo, Tadashi

2015-06-01

We review the current status of proteomics for esophageal cancer (EC) from a clinician's viewpoint. The ultimate goal of cancer proteomics is the improvement of clinical outcome. The proteome as a functional translation of the genome is a straightforward representation of genomic mechanisms that trigger carcinogenesis. Cancer proteomics has identified the mechanisms of carcinogenesis and tumor progression, detected biomarker candidates for early diagnosis, and provided novel therapeutic targets for personalized treatments. Our review focuses on three major topics in EC proteomics: diagnostics, treatment, and molecular mechanisms. We discuss the major histological differences between EC types, i.e., esophageal squamous cell carcinoma and adenocarcinoma, and evaluate the clinical significance of published proteomics studies, including promising diagnostic biomarkers and novel therapeutic targets, which should be further validated prior to launching clinical trials. Multi-disciplinary collaborations between basic scientists, clinicians, and pathologists should be established for inter-institutional validation. In conclusion, EC proteomics has provided significant results, which after thorough validation, should lead to the development of novel clinical tools and improvement of the clinical outcome for esophageal cancer patients. This article is part of a Special Issue entitled: Medical Proteomics. Copyright © 2014 Elsevier B.V. All rights reserved.

Omics on bioleaching: current and future impacts.

PubMed

Martinez, Patricio; Vera, Mario; Bobadilla-Fazzini, Roberto A

2015-10-01

Bioleaching corresponds to the microbial-catalyzed process of conversion of insoluble metals into soluble forms. As an applied biotechnology globally used, it represents an extremely interesting field of research where omics techniques can be applied in terms of knowledge development, but moreover in terms of process design, control, and optimization. In this mini-review, the current state of genomics, proteomics, and metabolomics of bioleaching and the major impacts of these analytical methods at industrial scale are highlighted. In summary, genomics has been essential in the determination of the biodiversity of leaching processes and for development of conceptual and functional metabolic models. Proteomic impacts are mostly related to microbe-mineral interaction analysis, including copper resistance and biofilm formation. Early steps of metabolomics in the field of bioleaching have shown a significant potential for the use of metabolites as industrial biomarkers. Development directions are given in order to enhance the future impacts of the omics in biohydrometallurgy.

The application of proteomics in different aspects of hepatocellular carcinoma research.

PubMed

Xing, Xiaohua; Liang, Dong; Huang, Yao; Zeng, Yongyi; Han, Xiao; Liu, Xiaolong; Liu, Jingfeng

2016-08-11

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, which is causing the second leading cancer-related death worldwide. With the significant advances of high-throughput protein analysis techniques, the proteomics offered an extremely useful and versatile analytical platform for biomedical researches. In recent years, different proteomic strategies have been widely applied in the various aspects of HCC studies, ranging from screening the early diagnostic and prognostic biomarkers to in-depth investigating the underlying molecular mechanisms. In this review, we would like to systematically summarize the current applications of proteomics in hepatocellular carcinoma study, and discuss the challenges of applying proteomics in study clinical samples, as well as discuss the possible application of proteomics in precision medicine. In this review, we have systematically summarized the current applications of proteomics in hepatocellular carcinoma study, ranging from screening biomarkers to in-depth investigating the underlying molecular mechanisms. In addition, we have discussed the challenges of applying proteomics in study clinical samples, as well as the possible applications of proteomics in precision medicine. We believe that this review would help readers to be better familiar with the recent progresses of clinical proteomics, especially in the field of hepatocellular carcinoma research. Copyright © 2016 Elsevier B.V. All rights reserved.

Affordable proteomics: the two-hybrid systems.

PubMed

Gillespie, Marc

2003-06-01

Numerous proteomic methodologies exist, but most require a heavy investment in expertise and technology. This puts these approaches out of reach for many laboratories and small companies, rarely allowing proteomics to be used as a pilot approach for biomarker or target identification. Two proteomic approaches, 2D gel electrophoresis and the two-hybrid systems, are currently available to most researchers. The two-hybrid systems, though accommodating to large-scale experiments, were originally designed as practical screens, that by comparison to current proteomics tools were small-scale, affordable and technically feasible. The screens rapidly generated data, identifying protein interactions that were previously uncharacterized. The foundation for a two-hybrid proteomic investigation can be purchased as separate kits from a number of companies. The true power of the technique lies not in its affordability, but rather in its portability. The two-hybrid system puts proteomics back into laboratories where the output of the screens can be evaluated by researchers with experience in the particular fields of basic research, cancer biology, toxicology or drug development.

Proteome of Caulobacter crescentus cell cycle publicly accessible on SWICZ server.

PubMed

Vohradsky, Jiri; Janda, Ivan; Grünenfelder, Björn; Berndt, Peter; Röder, Daniel; Langen, Hanno; Weiser, Jaroslav; Jenal, Urs

2003-10-01

Here we present the Swiss-Czech Proteomics Server (SWICZ), which hosts the proteomic database summarizing information about the cell cycle of the aquatic bacterium Caulobacter crescentus. The database provides a searchable tool for easy access of global protein synthesis and protein stability data as examined during the C. crescentus cell cycle. Protein synthesis data collected from five different cell cycle stages were determined for each protein spot as a relative value of the total amount of [(35)S]methionine incorporation. Protein stability of pulse-labeled extracts were measured during a chase period equivalent to one cell cycle unit. Quantitative information for individual proteins together with descriptive data such as protein identities, apparent molecular masses and isoelectric points, were combined with information on protein function, genomic context, and the cell cycle stage, and were then assembled in a relational database with a world wide web interface (http://proteom.biomed.cas.cz), which allows the database records to be searched and displays the recovered information. A total of 1250 protein spots were reproducibly detected on two-dimensional gel electropherograms, 295 of which were identified by mass spectroscopy. The database is accessible either through clickable two-dimensional gel electrophoretic maps or by means of a set of dedicated search engines. Basic characterization of the experimental procedures, data processing, and a comprehensive description of the web site are presented. In its current state, the SWICZ proteome database provides a platform for the incorporation of new data emerging from extended functional studies on the C. crescentus proteome.

Knowledge Translation: Moving Proteomics Science to Innovation in Society.

PubMed

Holmes, Christina; McDonald, Fiona; Jones, Mavis; Graham, Janice

2016-06-01

Proteomics is one of the pivotal next-generation biotechnologies in the current "postgenomics" era. Little is known about the ways in which innovative proteomics science is navigating the complex socio-political space between laboratory and society. It cannot be assumed that the trajectory between proteomics laboratory and society is linear and unidirectional. Concerned about public accountability and hopes for knowledge-based innovations, funding agencies and citizens increasingly expect that emerging science and technologies, such as proteomics, are effectively translated and disseminated as innovation in society. Here, we describe translation strategies promoted in the knowledge translation (KT) and science communication literatures and examine the use of these strategies within the field of proteomics. Drawing on data generated from qualitative interviews with proteomics scientists and ethnographic observation of international proteomics conferences over a 5-year period, we found that proteomics science incorporates a variety of KT strategies to reach knowledge users outside the field. To attain the full benefit of KT, however, proteomics scientists must challenge their own normative assumptions and approaches to innovation dissemination-beyond the current paradigm relying primarily on publication for one's scientific peers within one's field-and embrace the value of broader (interdisciplinary) KT strategies in promoting the uptake of their research. Notably, the Human Proteome Organization (HUPO) is paying increasing attention to a broader range of KT strategies, including targeted dissemination, integrated KT, and public outreach. We suggest that increasing the variety of KT strategies employed by proteomics scientists is timely and would serve well the omics system sciences community.

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

Developmental cigarette smoke exposure II: Hepatic proteome profiles in 6 month old adult offspring.

PubMed

Neal, Rachel E; Chen, Jing; Webb, Cindy; Stocke, Kendall; Gambrell, Caitlin; Greene, Robert M; Pisano, M Michele

2016-10-01

Utilizing a mouse model of 'active' developmental cigarette smoke exposure (CSE) [gestational day (GD) 1 through postnatal day (PD) 21] characterized by offspring low birth weight, the impact of developmental CSE on liver proteome profiles of adult offspring at 6 months of age was determined. Liver tissue was collected from Sham- and CSE-offspring for 2D-SDS-PAGE based proteome analysis with Partial Least Squares-Discriminant Analysis (PLS-DA). A similar study conducted at the cessation of exposure to cigarette smoke documented decreased gluconeogenesis coupled to oxidative stress in weanling offspring. In the current study, exposure throughout development to cigarette smoke resulted in impaired hepatic carbohydrate metabolism, decreased serum glucose levels, and increased gluconeogenic regulatory enzyme abundances during the fed-state coupled to decreased expression of SIRT1 as well as increased PEPCK and PGC1α expression. Together these findings indicate inappropriately timed gluconeogenesis that may reflect impaired insulin signaling in mature offspring exposed to 'active' developmental CSE. Copyright © 2016 Elsevier Inc. All rights reserved.

Proteomics of the Human Placenta: Promises and Realities

PubMed Central

Robinson, J.M.; Ackerman, W.E.; Kniss, D.A.; Takizawa, T.; Vandré, D.D.

2015-01-01

Proteomics is an area of study that sets as its ultimate goal the global analysis of all of the proteins expressed in a biological system of interest. However, technical limitations currently hamper proteome-wide analyses of complex systems. In a more practical sense, a desired outcome of proteomics research is the translation of large protein data sets into formats that provide meaningful information regarding clinical conditions (e.g., biomarkers to serve as diagnostic and/or prognostic indicators of disease). Herein, we discuss placental proteomics by describing existing studies, pointing out their strengths and weaknesses. In so doing, we strive to inform investigators interested in this area of research about the current gap between hyperbolic promises and realities. Additionally, we discuss the utility of proteomics in discovery-based research, particularly as regards the capacity to unearth novel insights into placental biology. Importantly, when considering under studied systems such as the human placenta and diseases associated with abnormalities in placental function, proteomics can serve as a robust ‘shortcut’ to obtaining information unlikely to be garnered using traditional approaches. PMID:18222537

Proteomics research in India: an update.

PubMed

Reddy, Panga Jaipal; Atak, Apurva; Ghantasala, Saicharan; Kumar, Saurabh; Gupta, Shabarni; Prasad, T S Keshava; Zingde, Surekha M; Srivastava, Sanjeeva

2015-09-08

After a successful completion of the Human Genome Project, deciphering the mystery surrounding the human proteome posed a major challenge. Despite not being largely involved in the Human Genome Project, the Indian scientific community contributed towards proteomic research along with the global community. Currently, more than 76 research/academic institutes and nearly 145 research labs are involved in core proteomic research across India. The Indian researchers have been major contributors in drafting the "human proteome map" along with international efforts. In addition to this, virtual proteomics labs, proteomics courses and remote triggered proteomics labs have helped to overcome the limitations of proteomics education posed due to expensive lab infrastructure. The establishment of Proteomics Society, India (PSI) has created a platform for the Indian proteomic researchers to share ideas, research collaborations and conduct annual conferences and workshops. Indian proteomic research is really moving forward with the global proteomics community in a quest to solve the mysteries of proteomics. A draft map of the human proteome enhances the enthusiasm among intellectuals to promote proteomic research in India to the world.This article is part of a Special Issue entitled: Proteomics in India. Copyright © 2015 Elsevier B.V. All rights reserved.

A decade of proteomics accomplished! Central and Eastern European Proteomic Conference (CEEPC) celebrates its 10th Anniversary in Budapest, Hungary.

PubMed

Gadher, Suresh Jivan; Drahos, László; Vékey, Károly; Kovarova, Hana

2017-07-01

The Central and Eastern European Proteomic Conference (CEEPC) proudly celebrated its 10th Anniversary with an exciting scientific program inclusive of proteome, proteomics and systems biology in Budapest, Hungary. Since 2007, CEEPC has represented 'state-of the-art' proteomics in and around Central and Eastern Europe and these series of conferences have become a well-recognized event in the proteomic calendar. Fresher challenges and global healthcare issues such as ageing and chronic diseases are driving clinical and scientific research towards regenerative, reparative and personalized medicine. To this end, proteomics may enable diverse intertwining research fields to reach their end goals. CEEPC will endeavor to facilitate these goals.

NIH Common Fund - Disruptive Proteomics Technologies - Challenges and Opportunities | Office of Cancer Clinical Proteomics Research

Cancer.gov

This Request for Information (RFI) is directed toward determining how best to accelerate research in disruptive proteomics technologies. The Disruptive Proteomics Technologies (DPT) Working Group of the NIH Common Fund wishes to identify gaps and opportunities in current technologies and methodologies related to proteome-wide measurements.  For the purposes of this RFI, “disruptive” is defined as very rapid, very significant gains, similar to the "disruptive" technology development that occurred in DNA sequencing technology.

A proteomic approach to obesity and type 2 diabetes

PubMed Central

López-Villar, Elena; Martos-Moreno, Gabriel Á; Chowen, Julie A; Okada, Shigeru; Kopchick, John J; Argente, Jesús

2015-01-01

The incidence of obesity and type diabetes 2 has increased dramatically resulting in an increased interest in its biomedical relevance. However, the mechanisms that trigger the development of diabetes type 2 in obese patients remain largely unknown. Scientific, clinical and pharmaceutical communities are dedicating vast resources to unravel this issue by applying different omics tools. During the last decade, the advances in proteomic approaches and the Human Proteome Organization have opened and are opening a new door that may be helpful in the identification of patients at risk and to improve current therapies. Here, we briefly review some of the advances in our understanding of type 2 diabetes that have occurred through the application of proteomics. We also review, in detail, the current improvements in proteomic methodologies and new strategies that could be employed to further advance our understanding of this pathology. By applying these new proteomic advances, novel therapeutic and/or diagnostic protein targets will be discovered in the obesity/Type 2 diabetes area. PMID:25960181

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

Comparative proteome analysis of Milnesium tardigradum in early embryonic state versus adults in active and anhydrobiotic state.

PubMed

Schokraie, Elham; Warnken, Uwe; Hotz-Wagenblatt, Agnes; Grohme, Markus A; Hengherr, Steffen; Förster, Frank; Schill, Ralph O; Frohme, Marcus; Dandekar, Thomas; Schnölzer, Martina

2012-01-01

Tardigrades have fascinated researchers for more than 300 years because of their extraordinary capability to undergo cryptobiosis and survive extreme environmental conditions. However, the survival mechanisms of tardigrades are still poorly understood mainly due to the absence of detailed knowledge about the proteome and genome of these organisms. Our study was intended to provide a basis for the functional characterization of expressed proteins in different states of tardigrades. High-throughput, high-accuracy proteomics in combination with a newly developed tardigrade specific protein database resulted in the identification of more than 3000 proteins in three different states: early embryonic state and adult animals in active and anhydrobiotic state. This comprehensive proteome resource includes protein families such as chaperones, antioxidants, ribosomal proteins, cytoskeletal proteins, transporters, protein channels, nutrient reservoirs, and developmental proteins. A comparative analysis of protein families in the different states was performed by calculating the exponentially modified protein abundance index which classifies proteins in major and minor components. This is the first step to analyzing the proteins involved in early embryonic development, and furthermore proteins which might play an important role in the transition into the anhydrobiotic state.

Comparative proteome analysis of Milnesium tardigradum in early embryonic state versus adults in active and anhydrobiotic state

PubMed Central

Schokraie, Elham; Warnken, Uwe; Hotz-Wagenblatt, Agnes; Grohme, Markus A.; Hengherr, Steffen; Förster, Frank; Schill, Ralph O.; Frohme, Marcus; Dandekar, Thomas; Schnölzer, Martina

2012-01-01

Tardigrades have fascinated researchers for more than 300 years because of their extraordinary capability to undergo cryptobiosis and survive extreme environmental conditions. However, the survival mechanisms of tardigrades are still poorly understood mainly due to the absence of detailed knowledge about the proteome and genome of these organisms. Our study was intended to provide a basis for the functional characterization of expressed proteins in different states of tardigrades. High-throughput, high-accuracy proteomics in combination with a newly developed tardigrade specific protein database resulted in the identification of more than 3000 proteins in three different states: early embryonic state and adult animals in active and anhydrobiotic state. This comprehensive proteome resource includes protein families such as chaperones, antioxidants, ribosomal proteins, cytoskeletal proteins, transporters, protein channels, nutrient reservoirs, and developmental proteins. A comparative analysis of protein families in the different states was performed by calculating the exponentially modified protein abundance index which classifies proteins in major and minor components. This is the first step to analyzing the proteins involved in early embryonic development, and furthermore proteins which might play an important role in the transition into the anhydrobiotic state. PMID:23029181

Respiratory Proteomics Today: Are Technological Advances for the Identification of Biomarker Signatures Catching up with Their Promise? A Critical Review of the Literature in the Decade 2004-2013.

PubMed

Viglio, Simona; Stolk, Jan; Iadarola, Paolo; Giuliano, Serena; Luisetti, Maurizio; Salvini, Roberta; Fumagalli, Marco; Bardoni, Anna

2014-01-22

To improve the knowledge on a variety of severe disorders, research has moved from the analysis of individual proteins to the investigation of all proteins expressed by a tissue/organism. This global proteomic approach could prove very useful: (i) for investigating the biochemical pathways involved in disease; (ii) for generating hypotheses; or (iii) as a tool for the identification of proteins differentially expressed in response to the disease state. Proteomics has not been used yet in the field of respiratory research as extensively as in other fields, only a few reproducible and clinically applicable molecular markers, which can assist in diagnosis, having been currently identified. The continuous advances in both instrumentation and methodology, which enable sensitive and quantitative proteomic analyses in much smaller amounts of biological material than before, will hopefully promote the identification of new candidate biomarkers in this area. The aim of this report is to critically review the application over the decade 2004-2013 of very sophisticated technologies to the study of respiratory disorders. The observed changes in protein expression profiles from tissues/fluids of patients affected by pulmonary disorders opens the route for the identification of novel pathological mediators of these disorders.

Murine colon proteome and characterization of the protein pathways

PubMed Central

2012-01-01

Background Most of the current proteomic researches focus on proteome alteration due to pathological disorders (i.e.: colorectal cancer) rather than normal healthy state when mentioning colon. As a result, there are lacks of information regarding normal whole tissue- colon proteome. Results We report here a detailed murine (mouse) whole tissue- colon protein reference dataset composed of 1237 confident protein (FDR < 2) with comprehensive insight on its peptide properties, cellular and subcellular localization, functional network GO annotation analysis, and its relative abundances. The presented dataset includes wide spectra of pI and Mw ranged from 3–12 and 4–600 KDa, respectively. Gravy index scoring predicted 19.5% membranous and 80.5% globularly located proteins. GO hierarchies and functional network analysis illustrated proteins function together with their relevance and implication of several candidates in malignancy such as Mitogen- activated protein kinase (Mapk8, 9) in colorectal cancer, Fibroblast growth factor receptor (Fgfr 2), Glutathione S-transferase (Gstp1) in prostate cancer, and Cell division control protein (Cdc42), Ras-related protein (Rac1,2) in pancreatic cancer. Protein abundances calculated with 3 different algorithms (NSAF, PAF and emPAI) provide a relative quantification under normal condition as guidance. Conclusions This highly confidence colon proteome catalogue will not only serve as a useful reference for further experiments characterizing differentially expressed proteins induced from diseased conditions, but also will aid in better understanding the ontology and functional absorptive mechanism of the colon as well. PMID:22929016

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

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, ...

Liquid Chromatography-Mass Spectrometry-based Quantitative Proteomics

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

Xie, Fang; Liu, Tao; Qian, Weijun

2011-07-22

Liquid chromatography-mass spectrometry (LC-MS)-based quantitative proteomics has become increasingly applied for a broad range of biological applications due to growing capabilities for broad proteome coverage and good accuracy in quantification. Herein, we review the current LC-MS-based quantification methods with respect to their advantages and limitations, and highlight their potential applications.

A proteomic approach to obesity and type 2 diabetes.

PubMed

López-Villar, Elena; Martos-Moreno, Gabriel Á; Chowen, Julie A; Okada, Shigeru; Kopchick, John J; Argente, Jesús

2015-07-01

The incidence of obesity and type diabetes 2 has increased dramatically resulting in an increased interest in its biomedical relevance. However, the mechanisms that trigger the development of diabetes type 2 in obese patients remain largely unknown. Scientific, clinical and pharmaceutical communities are dedicating vast resources to unravel this issue by applying different omics tools. During the last decade, the advances in proteomic approaches and the Human Proteome Organization have opened and are opening a new door that may be helpful in the identification of patients at risk and to improve current therapies. Here, we briefly review some of the advances in our understanding of type 2 diabetes that have occurred through the application of proteomics. We also review, in detail, the current improvements in proteomic methodologies and new strategies that could be employed to further advance our understanding of this pathology. By applying these new proteomic advances, novel therapeutic and/or diagnostic protein targets will be discovered in the obesity/Type 2 diabetes area. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Proteomics Analysis of the Causative Agent of Typhoid Fever

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

Ansong, Charles; Yoon, Hyunjin; Norbeck, Angela D.

2008-02-01

Typhoid fever is a potentially fatal disease caused by the bacterial pathogen Salmonella enterica serovar Typhi (S. typhi). S. typhi infection is a complex process that involves numerous bacterially-encoded virulence determinants, and these are thought to confer both stringent human host specificity and a high mortality rate. In the present study we used a liquid chromatography-mass spectrometry (LC-MS) based proteomics strategy to investigate the proteome of logarithmic, stationary phase, and low pH/low magnesium (MgM) S. typhi cultures. This represents the first large scale comprehensive characterization of the S. typhi proteome. Our analysis identified a total of 2066 S. typhi proteins.more » In an effort to identify putative S. typhi-specific virulence factors, we then compared our S. typhi results to those obtained in a previously published study of the S. typhimurium proteome under similar conditions (Adkins J.N. et al (2006) Mol Cell Prot). Comparative proteomic analysis of S. typhi (strain Ty2) and S. typhimurium (strains LT2 and 14028) revealed a subset of highly expressed proteins unique to S. typhi that were exclusively detected under conditions that mimic the infective state in macrophage cells. These proteins included CdtB, HlyE, and a conserved protein encoded by t1476. The differential expression of selected proteins was confirmed by Western blot analysis. Taken together with the current literature, our observations suggest that this subset of proteins may play a role in S. typhi pathogenesis and human host specificity. In addition, we observed products of the biotin (bio) operon displayed a higher abundance in the more virulent strains S. typhi-Ty2 and S. typhimurium-14028 compared to the virulence attenuated S. typhimurium strain LT2, suggesting bio proteins may contribute to Salmonella pathogenesis.« less

Aptamer-based multiplexed proteomic technology for biomarker discovery.

PubMed

Gold, Larry; Ayers, Deborah; Bertino, Jennifer; Bock, Christopher; Bock, Ashley; Brody, Edward N; Carter, Jeff; Dalby, Andrew B; Eaton, Bruce E; Fitzwater, Tim; Flather, Dylan; Forbes, Ashley; Foreman, Trudi; Fowler, Cate; Gawande, Bharat; Goss, Meredith; Gunn, Magda; Gupta, Shashi; Halladay, Dennis; Heil, Jim; Heilig, Joe; Hicke, Brian; Husar, Gregory; Janjic, Nebojsa; Jarvis, Thale; Jennings, Susan; Katilius, Evaldas; Keeney, Tracy R; Kim, Nancy; Koch, Tad H; Kraemer, Stephan; Kroiss, Luke; Le, Ngan; Levine, Daniel; Lindsey, Wes; Lollo, Bridget; Mayfield, Wes; Mehan, Mike; Mehler, Robert; Nelson, Sally K; Nelson, Michele; Nieuwlandt, Dan; Nikrad, Malti; Ochsner, Urs; Ostroff, Rachel M; Otis, Matt; Parker, Thomas; Pietrasiewicz, Steve; Resnicow, Daniel I; Rohloff, John; Sanders, Glenn; Sattin, Sarah; Schneider, Daniel; Singer, Britta; Stanton, Martin; Sterkel, Alana; Stewart, Alex; Stratford, Suzanne; Vaught, Jonathan D; Vrkljan, Mike; Walker, Jeffrey J; Watrobka, Mike; Waugh, Sheela; Weiss, Allison; Wilcox, Sheri K; Wolfson, Alexey; Wolk, Steven K; Zhang, Chi; Zichi, Dom

2010-12-07

The interrogation of proteomes ("proteomics") in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (~100 fM-1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states. We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.

The state of proteome profiling in the fungal genus Aspergillus.

PubMed

Kim, Yonghyun; Nandakumar, M P; Marten, Mark R

2008-03-01

Aspergilli are an important genus of filamentous fungi that contribute to a multibillion dollar industry. Since many fungal genome sequencing were recently completed, it would be advantageous to profile their proteome to better understand the fungal cell factory. Here, we review proteomic data generated for the Aspergilli in recent years. Thus far, a combined total of 28 cell surface, 102 secreted and 139 intracellular proteins have been identified based on 10 different studies on Aspergillus proteomics. A summary proteome map highlighting identified proteins in major metabolic pathway is presented.

Integrated metabolomics and proteomics highlight altered nicotinamide and polyamine pathways in lung adenocarcinoma

PubMed Central

Fahrmann, Johannes F.; Grapov, Dmitry; Wanichthanarak, Kwanjeera; DeFelice, Brian C.; Salemi, Michelle R.; Rom, William N.; Gandara, David R.; Phinney, Brett S.; Fiehn, Oliver; Pass, Harvey

2017-01-01

Abstract Lung cancer is the leading cause of cancer mortality in the United States with non-small cell lung cancer adenocarcinoma being the most common histological type. Early perturbations in cellular metabolism are a hallmark of cancer, but the extent of these changes in early stage lung adenocarcinoma remains largely unknown. In the current study, an integrated metabolomics and proteomics approach was utilized to characterize the biochemical and molecular alterations between malignant and matched control tissue from 27 subjects diagnosed with early stage lung adenocarcinoma. Differential analysis identified 71 metabolites and 1102 proteins that delineated tumor from control tissue. Integrated results indicated four major metabolic changes in early stage adenocarcinoma (1): increased glycosylation and glutaminolysis (2); elevated Nrf2 activation (3); increase in nicotinic and nicotinamide salvaging pathways and (4) elevated polyamine biosynthesis linked to differential regulation of the s-adenosylmethionine/nicotinamide methyl-donor pathway. Genomic data from publicly available databases were included to strengthen proteomic findings. Our findings provide insight into the biochemical and molecular biological reprogramming that may accompany early stage lung tumorigenesis and highlight potential therapeutic targets. PMID:28049629

Mass spectrometric identification of proteins in complex post-genomic projects. Soluble proteins of the metabolically versatile, denitrifying 'Aromatoleum' sp. strain EbN1.

PubMed

Hufnagel, Peter; Rabus, Ralf

2006-01-01

The rapidly developing proteomics technologies help to advance the global understanding of physiological and cellular processes. The lifestyle of a study organism determines the type and complexity of a given proteomic project. The complexity of this study is characterized by a broad collection of pathway-specific subproteomes, reflecting the metabolic versatility as well as the regulatory potential of the aromatic-degrading, denitrifying bacterium 'Aromatoleum' sp. strain EbN1. Differences in protein profiles were determined using a gel-based approach. Protein identification was based on a progressive application of MALDI-TOF-MS, MALDI-TOF-MS/MS and LC-ESI-MS/MS. This progression was result-driven and automated by software control. The identification rate was increased by the assembly of a project-specific list of background signals that was used for internal calibration of the MS spectra, and by the combination of two search engines using a dedicated MetaScoring algorithm. In total, intelligent bioinformatics could increase the identification yield from 53 to 70% of the analyzed 5,050 gel spots; a total of 556 different proteins were identified. MS identification was highly reproducible: most proteins were identified more than twice from parallel 2DE gels with an average sequence coverage of >50% and rather restrictive score thresholds (Mascot >or=95, ProFound >or=2.2, MetaScore >or=97). The MS technologies and bioinformatics tools that were implemented and integrated to handle this complex proteomic project are presented. In addition, we describe the basic principles and current developments of the applied technologies and provide an overview over the current state of microbial proteome research. Copyright (c) 2006 S. Karger AG, Basel.

Proteomics Standards Initiative: Fifteen Years of Progress and Future Work

PubMed Central

2017-01-01

The Proteomics Standards Initiative (PSI) of the Human Proteome Organization (HUPO) has now been developing and promoting open community standards and software tools in the field of proteomics for 15 years. Under the guidance of the chair, cochairs, and other leadership positions, the PSI working groups are tasked with the development and maintenance of community standards via special workshops and ongoing work. Among the existing ratified standards, the PSI working groups continue to update PSI-MI XML, MITAB, mzML, mzIdentML, mzQuantML, mzTab, and the MIAPE (Minimum Information About a Proteomics Experiment) guidelines with the advance of new technologies and techniques. Furthermore, new standards are currently either in the final stages of completion (proBed and proBAM for proteogenomics results as well as PEFF) or in early stages of design (a spectral library standard format, a universal spectrum identifier, the qcML quality control format, and the Protein Expression Interface (PROXI) web services Application Programming Interface). In this work we review the current status of all of these aspects of the PSI, describe synergies with other efforts such as the ProteomeXchange Consortium, the Human Proteome Project, and the metabolomics community, and provide a look at future directions of the PSI. PMID:28849660

Proteomics Standards Initiative: Fifteen Years of Progress and Future Work.

PubMed

Deutsch, Eric W; Orchard, Sandra; Binz, Pierre-Alain; Bittremieux, Wout; Eisenacher, Martin; Hermjakob, Henning; Kawano, Shin; Lam, Henry; Mayer, Gerhard; Menschaert, Gerben; Perez-Riverol, Yasset; Salek, Reza M; Tabb, David L; Tenzer, Stefan; Vizcaíno, Juan Antonio; Walzer, Mathias; Jones, Andrew R

2017-12-01

The Proteomics Standards Initiative (PSI) of the Human Proteome Organization (HUPO) has now been developing and promoting open community standards and software tools in the field of proteomics for 15 years. Under the guidance of the chair, cochairs, and other leadership positions, the PSI working groups are tasked with the development and maintenance of community standards via special workshops and ongoing work. Among the existing ratified standards, the PSI working groups continue to update PSI-MI XML, MITAB, mzML, mzIdentML, mzQuantML, mzTab, and the MIAPE (Minimum Information About a Proteomics Experiment) guidelines with the advance of new technologies and techniques. Furthermore, new standards are currently either in the final stages of completion (proBed and proBAM for proteogenomics results as well as PEFF) or in early stages of design (a spectral library standard format, a universal spectrum identifier, the qcML quality control format, and the Protein Expression Interface (PROXI) web services Application Programming Interface). In this work we review the current status of all of these aspects of the PSI, describe synergies with other efforts such as the ProteomeXchange Consortium, the Human Proteome Project, and the metabolomics community, and provide a look at future directions of the PSI.

Accurate, Sensitive, and Precise Multiplexed Proteomics Using the Complement Reporter Ion Cluster

DOE PAGES

Sonnett, Matthew; Yeung, Eyan; Wuhr, Martin

2018-03-09

We present that quantitative analysis of proteomes across multiple time points, organelles, and perturbations is essential for understanding both fundamental biology and disease states. The development of isobaric tags (e.g. TMT) have enabled the simultaneous measurement of peptide abundances across several different conditions. These multiplexed approaches are promising in principle because of advantages in throughput and measurement quality. However, in practice existing multiplexing approaches suffer from key limitations. In its simple implementation (TMT-MS2), measurements are distorted by chemical noise leading to poor measurement accuracy. The current state-of-the-art (TMT-MS3) addresses this, but requires specialized quadrupole-iontrap-Orbitrap instrumentation. The complement reporter ion approachmore » (TMTc) produces high accuracy measurements and is compatible with many more instruments, like quadrupole-Orbitraps. However, the required deconvolution of the TMTc cluster leads to poor measurement precision. Here, we introduce TMTc+, which adds the modeling of the MS2-isolation step into the deconvolution algorithm. The resulting measurements are comparable in precision to TMT-MS3/MS2. The improved duty cycle, and lower filtering requirements make TMTc+ more sensitive than TMT-MS3 and comparable with TMT-MS2. At the same time, unlike TMT-MS2, TMTc+ is exquisitely able to distinguish signal from chemical noise even outperforming TMT-MS3. Lastly, we compare TMTc+ to quantitative label-free proteomics of total HeLa lysate and find that TMTc+ quantifies 7.8k versus 3.9k proteins in a 5-plex sample. At the same time the median coefficient of variation improves from 13% to 4%. Furthermore, TMTc+ advances quantitative proteomics by enabling accurate, sensitive, and precise multiplexed experiments on more commonly used instruments.« less

Accurate, Sensitive, and Precise Multiplexed Proteomics Using the Complement Reporter Ion Cluster

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

Sonnett, Matthew; Yeung, Eyan; Wuhr, Martin

We present that quantitative analysis of proteomes across multiple time points, organelles, and perturbations is essential for understanding both fundamental biology and disease states. The development of isobaric tags (e.g. TMT) have enabled the simultaneous measurement of peptide abundances across several different conditions. These multiplexed approaches are promising in principle because of advantages in throughput and measurement quality. However, in practice existing multiplexing approaches suffer from key limitations. In its simple implementation (TMT-MS2), measurements are distorted by chemical noise leading to poor measurement accuracy. The current state-of-the-art (TMT-MS3) addresses this, but requires specialized quadrupole-iontrap-Orbitrap instrumentation. The complement reporter ion approachmore » (TMTc) produces high accuracy measurements and is compatible with many more instruments, like quadrupole-Orbitraps. However, the required deconvolution of the TMTc cluster leads to poor measurement precision. Here, we introduce TMTc+, which adds the modeling of the MS2-isolation step into the deconvolution algorithm. The resulting measurements are comparable in precision to TMT-MS3/MS2. The improved duty cycle, and lower filtering requirements make TMTc+ more sensitive than TMT-MS3 and comparable with TMT-MS2. At the same time, unlike TMT-MS2, TMTc+ is exquisitely able to distinguish signal from chemical noise even outperforming TMT-MS3. Lastly, we compare TMTc+ to quantitative label-free proteomics of total HeLa lysate and find that TMTc+ quantifies 7.8k versus 3.9k proteins in a 5-plex sample. At the same time the median coefficient of variation improves from 13% to 4%. Furthermore, TMTc+ advances quantitative proteomics by enabling accurate, sensitive, and precise multiplexed experiments on more commonly used instruments.« less

Integrated redox proteomics and metabolomics of mitochondria to identify mechanisms of cd toxicity.

PubMed

Go, Young-Mi; Roede, James R; Orr, Michael; Liang, Yongliang; Jones, Dean P

2014-05-01

Cadmium (Cd) exposure contributes to human diseases affecting liver, kidney, lung, and other organ systems, but mechanisms underlying the pleotropic nature of these toxicities are poorly understood. Cd accumulates in humans from dietary, environmental (including cigarette smoke), and occupational sources, and has a twenty-year biologic half-life. Our previous mouse and cell studies showed that environmental low-dose Cd exposure altered protein redox states resulting in stimulation of inflammatory signaling and disruption of the actin cytoskeleton system, suggesting that Cd could impact multiple mechanisms of disease. In the current study, we investigated the effects of acute Cd exposure on the redox proteome and metabolome of mouse liver mitochondria to gain insight into associated toxicological mechanisms and functions. We analyzed redox states of liver mitochondrial proteins by redox proteomics using isotope coded affinity tag (ICAT) combined mass spectrometry. Redox ICAT identified 2687 cysteine-containing peptides (peptidyl Cys) of which 1667 peptidyl Cys (657 proteins) were detected in both control and Cd-exposed samples. Of these, 46% (1247 peptidyl Cys, 547 proteins) were oxidized by Cd more than 1.5-fold relative to controls. Bioinformatics analysis using MetaCore software showed that Cd affected 86 pathways, including 24 Cys in proteins functioning in branched chain amino acid (BCAA) and 14 Cys in proteins functioning in fatty acid (acylcarnitine/carnitine) metabolism. Consistently, high-resolution metabolomics data showed that Cd treatment altered levels of BCAA and carnitine metabolites. Together, these results show that mitochondrial protein redox and metabolites are targets in Cd-induced hepatotoxicity. The results further indicate that redox proteomics and metabolomics can be used in an integrated systems approach to investigate complex disease mechanisms.

Quantitative proteomics in the field of microbiology.

PubMed

Otto, Andreas; Becher, Dörte; Schmidt, Frank

2014-03-01

Quantitative proteomics has become an indispensable analytical tool for microbial research. Modern microbial proteomics covers a wide range of topics in basic and applied research from in vitro characterization of single organisms to unravel the physiological implications of stress/starvation to description of the proteome content of a cell at a given time. With the techniques available, ranging from classical gel-based procedures to modern MS-based quantitative techniques, including metabolic and chemical labeling, as well as label-free techniques, quantitative proteomics is today highly successful in sophisticated settings of high complexity such as host-pathogen interactions, mixed microbial communities, and microbial metaproteomics. In this review, we will focus on the vast range of techniques practically applied in current research with an introduction of the workflows used for quantitative comparisons, a description of the advantages/disadvantages of the various methods, reference to hallmark publications and presentation of applications in current microbial research. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Progress and pitfalls in finding the 'missing proteins' from the human proteome map.

PubMed

Segura, Victor; Garin-Muga, Alba; Guruceaga, Elizabeth; Corrales, Fernando J

2017-01-01

The Human Proteome Project was launched with two main goals: the comprehensive and systematic definition of the human proteome map and the development of ready to use analytical tools to measure relevant proteins in their biological context in health and disease. Despite the great progress in this endeavour, there is still a group of reluctant proteins with no, or scarce, experimental evidence supporting their existence. These are called the 'missing proteins' and represent one of the biggest challenges to complete the human proteome map. Areas covered: This review focuses on the description of the missing proteome based on the HUPO standards, the analysis of the reasons explaining the difficulty of detecting missing proteins and the strategies currently used in the search for missing proteins. The present and future of the quest for the missing proteins is critically revised hereafter. Expert commentary: An overarching multidisciplinary effort is currently being done under the HUPO umbrella to allow completion of the human proteome map. It is expected that the detection of missing proteins will grow in the coming years since the methods and the best tissue/cell type sample for their search are already on the table.

Five years later: the current status of the use of proteomics and transcriptomics in EMF research.

PubMed

Leszczynski, Dariusz; de Pomerai, David; Koczan, Dirk; Stoll, Dieter; Franke, Helmut; Albar, Juan Pablo

2012-08-01

The World Health Organization's and Radiation and Nuclear Safety Authority's "Workshop on Application of Proteomics and Transcriptomics in Electromagnetic Fields Research" was held in Helsinki in the October/November 2005. As a consequence of this meeting, Proteomics journal published in 2006 a special issue "Application of Proteomics and Transcriptomics in EMF Research" (Vol. 6 No. 17; Guest Editor: D. Leszczynski). This Proteomics issue presented the status of research, of the effects of electromagnetic fields (EMF) using proteomics and transcriptomics methods, present in 2005. The current overview/opinion article presents the status of research in this area by reviewing all studies that were published by the end of 2010. The review work was a part of the European Cooperation in the Field of Scientific and Technical Research (COST) Action BM0704 that created a structure in which researchers in the field of EMF and health shared knowledge and information. The review was prepared by the members of the COST Action BM0704 task group on the high-throughput screening techniques and electromagnetic fields (TG-HTST-EMF). © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microfluidic-Mass Spectrometry Interfaces for Translational Proteomics.

PubMed

Pedde, R Daniel; Li, Huiyan; Borchers, Christoph H; Akbari, Mohsen

2017-10-01

Interfacing mass spectrometry (MS) with microfluidic chips (μchip-MS) holds considerable potential to transform a clinician's toolbox, providing translatable methods for the early detection, diagnosis, monitoring, and treatment of noncommunicable diseases by streamlining and integrating laborious sample preparation workflows on high-throughput, user-friendly platforms. Overcoming the limitations of competitive immunoassays - currently the gold standard in clinical proteomics - μchip-MS can provide unprecedented access to complex proteomic assays having high sensitivity and specificity, but without the labor, costs, and complexities associated with conventional MS sample processing. This review surveys recent μchip-MS systems for clinical applications and examines their emerging role in streamlining the development and translation of MS-based proteomic assays by alleviating many of the challenges that currently inhibit widespread clinical adoption. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Cell wall proteome of pathogenic fungi.

PubMed

Karkowska-Kuleta, Justyna; Kozik, Andrzej

2015-01-01

A fast development of a wide variety of proteomic techniques supported by mass spectrometry coupled with high performance liquid chromatography has been observed in recent years. It significantly contributes to the progress in research on the cell wall, very important part of the cells of pathogenic fungi. This complicated structure composed of different polysaccharides, proteins, lipids and melanin, plays a key role in interactions with the host during infection. Changes in the set of the surface-exposed proteins under different environmental conditions provide an effective way for pathogens to respond, adapt and survive in the new niches of infection. This work summarizes the current state of knowledge on proteins, studied both qualitatively and quantitatively, and found within the cell wall of fungal pathogens for humans, including Candida albicans, Candida glabrata, Aspergillus fumigatus, Cryptococcus neoformans and other medically important fungi. The described proteomic studies involved the isolation and fractionation of particular sets of proteins of interest with various techniques, often based on differences in their linkages to the polysaccharide scaffold. Furthermore, the proteinaceous contents of extracellular vesicles ("virulence bags") of C. albicans, C. neoformans, Histoplasma capsulatum and Paracoccidioides brasiliensis are compared, because their production can partially explain the problem of non-classical protein secretion by fungi. The role assigned to surface-exposed proteins in pathogenesis of fungal infections is enormously high, thus justifying the need for further investigation of cell wall proteomes.

Proteomic landscape in Central and Eastern Europe: the 9th Central and Eastern European Proteomic Conference, Poznań, Poland.

PubMed

Gadher, Suresh Jivan; Marczak, Łukasz; Łuczak, Magdalena; Stobiecki, Maciej; Widlak, Piotr; Kovarova, Hana

2016-01-01

Every year since 2007, the Central and Eastern European Proteomic Conference (CEEPC) has excelled in representing state-of-the-art proteomics in and around Central and Eastern Europe, and linking it to international institutions worldwide. Its mission remains to contribute to all approaches of proteomics including traditional and often-revisited methodologies as well as the latest technological achievements in clinical, quantitative and structural proteomics with a view to systems biology of a variety of processes. The 9th CEEPC was held from June 15th to 18th, 2015, at the Institute of Bioorganic Chemistry, Polish Academy of Sciences in Poznań, Poland. The scientific program stimulated exchange of proteomic knowledge whilst the spectacular venue of the conference allowed participants to enjoy the cobblestoned historical city of Poznań.

Microgravity-driven remodeling of the proteome reveals insights into molecular mechanisms and signal networks involved in response to the space flight environment.

PubMed

Rea, Giuseppina; Cristofaro, Francesco; Pani, Giuseppe; Pascucci, Barbara; Ghuge, Sandip A; Corsetto, Paola Antonia; Imbriani, Marcello; Visai, Livia; Rizzo, Angela M

2016-03-30

Space is a hostile environment characterized by high vacuum, extreme temperatures, meteoroids, space debris, ionospheric plasma, microgravity and space radiation, which all represent risks for human health. A deep understanding of the biological consequences of exposure to the space environment is required to design efficient countermeasures to minimize their negative impact on human health. Recently, proteomic approaches have received a significant amount of attention in the effort to further study microgravity-induced physiological changes. In this review, we summarize the current knowledge about the effects of microgravity on microorganisms (in particular Cupriavidus metallidurans CH34, Bacillus cereus and Rhodospirillum rubrum S1H), plants (whole plants, organs, and cell cultures), mammalian cells (endothelial cells, bone cells, chondrocytes, muscle cells, thyroid cancer cells, immune system cells) and animals (invertebrates, vertebrates and mammals). Herein, we describe their proteome's response to microgravity, focusing on proteomic discoveries and their future potential applications in space research. Space experiments and operational flight experience have identified detrimental effects on human health and performance because of exposure to weightlessness, even when currently available countermeasures are implemented. Many experimental tools and methods have been developed to study microgravity induced physiological changes. Recently, genomic and proteomic approaches have received a significant amount of attention. This review summarizes the recent research studies of the proteome response to microgravity inmicroorganisms, plants, mammalians cells and animals. Current proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of all proteomes. Understanding gene and/or protein expression is the key to unlocking the mechanisms behind microgravity-induced problems and to finding effective countermeasures to spaceflight-induced alterations but also for the study of diseases on earth. Future perspectives are also highlighted. Copyright © 2015 Elsevier B.V. All rights reserved.

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 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

CPTAC Launches Proteomics Data Portal | Office of Cancer Clinical Proteomics Research

Cancer.gov

The National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) announces the launch of the CPTAC Data Portal. The Data Portal hosts all the data that is currently being produced by the consortium with additional historic data from CPTAC 1. The total amount of hosted data exceeds over 500 GB of RAW data in over 800 files.

Recent advances in applying mass spectrometry and systems biology to determine brain dynamics.

PubMed

Scifo, Enzo; Calza, Giulio; Fuhrmann, Martin; Soliymani, Rabah; Baumann, Marc; Lalowski, Maciej

2017-06-01

Neurological disorders encompass various pathologies which disrupt normal brain physiology and function. Poor understanding of their underlying molecular mechanisms and their societal burden argues for the necessity of novel prevention strategies, early diagnostic techniques and alternative treatment options to reduce the scale of their expected increase. Areas covered: This review scrutinizes mass spectrometry based approaches used to investigate brain dynamics in various conditions, including neurodegenerative and neuropsychiatric disorders. Different proteomics workflows for isolation/enrichment of specific cell populations or brain regions, sample processing; mass spectrometry technologies, for differential proteome quantitation, analysis of post-translational modifications and imaging approaches in the brain are critically deliberated. Future directions, including analysis of cellular sub-compartments, targeted MS platforms (selected/parallel reaction monitoring) and use of mass cytometry are also discussed. Expert commentary: Here, we summarize and evaluate current mass spectrometry based approaches for determining brain dynamics in health and diseases states, with a focus on neurological disorders. Furthermore, we provide insight on current trends and new MS technologies with potential to improve this analysis.

HaloTag Technology: A Versatile Platform for Biomedical Applications

PubMed Central

2015-01-01

Exploration of protein function and interaction is critical for discovering links among genomics, proteomics, and disease state; yet, the immense complexity of proteomics found in biological systems currently limits our investigational capacity. Although affinity and autofluorescent tags are widely employed for protein analysis, these methods have been met with limited success because they lack specificity and require multiple fusion tags and genetic constructs. As an alternative approach, the innovative HaloTag protein fusion platform allows protein function and interaction to be comprehensively analyzed using a single genetic construct with multiple capabilities. This is accomplished using a simplified process, in which a variable HaloTag ligand binds rapidly to the HaloTag protein (usually linked to the protein of interest) with high affinity and specificity. In this review, we examine all current applications of the HaloTag technology platform for biomedical applications, such as the study of protein isolation and purification, protein function, protein–protein and protein–DNA interactions, biological assays, in vitro cellular imaging, and in vivo molecular imaging. In addition, novel uses of the HaloTag platform are briefly discussed along with potential future applications. PMID:25974629

Advances in microscale separations towards nanoproteomics applications

DOE PAGES

Yi, Lian; Piehowski, Paul D.; Shi, Tujin; ...

2017-07-21

Microscale separation (e.g., liquid chromatography or capillary electrophoresis) coupled with mass spectrometry (MS) has become the primary tool for advanced proteomics, an indispensable technology for gaining understanding of complex biological processes. In recent decades significant advances have been achieved in MS-based proteomics. But, the current proteomics platforms still face an analytical challenge in overall sensitivity towards nanoproteomics applications for starting materials of less than 1 μg total proteins (e.g., cellular heterogeneity in tissue pathologies). We review recent advances in microscale separation techniques and integrated sample processing strategies that improve the overall sensitivity and proteome coverage of the proteomics workflow, andmore » their contributions towards nanoproteomics applications.« less

Advances in microscale separations towards nanoproteomics applications

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

Yi, Lian; Piehowski, Paul D.; Shi, Tujin

Microscale separation (e.g., liquid chromatography or capillary electrophoresis) coupled with mass spectrometry (MS) has become the primary tool for advanced proteomics, an indispensable technology for gaining understanding of complex biological processes. In recent decades significant advances have been achieved in MS-based proteomics. But, the current proteomics platforms still face an analytical challenge in overall sensitivity towards nanoproteomics applications for starting materials of less than 1 μg total proteins (e.g., cellular heterogeneity in tissue pathologies). We review recent advances in microscale separation techniques and integrated sample processing strategies that improve the overall sensitivity and proteome coverage of the proteomics workflow, andmore » their contributions towards nanoproteomics applications.« less

CPTAC Teams | Office of Cancer Clinical Proteomics Research

Cancer.gov

The following are the current CPTAC teams, representing a network of Proteome Characterization Centers (PCCs), Proteogenomic Translational Research Centers (PTRCs), and Proteogenomic Data Analysis Centers (PGDACs). Teams are listed alphabetically by institution, with their respective Principal Investigators:

Advances in Proteomics of Mycobacterium leprae.

PubMed

Parkash, O; Singh, B P

2012-04-01

Although Mycobacterium leprae was the first bacterial pathogen identified causing human disease, it remains one of the few that is non-cultivable. Understanding the biology of M. leprae is one of the primary challenges in current leprosy research. Genomics has been extremely valuable, nonetheless, functional proteins are ultimately responsible for controlling most aspects of cellular functions, which in turn could facilitate parasitizing the host. Furthermore, bacterial proteins provide targets for most of the vaccines and immunodiagnostic tools. Better understanding of the proteomics of M. leprae could also help in developing new drugs against M. leprae. During the past nearly 15 years, there have been several developments towards the identification of M. leprae proteins employing contemporary proteomics tools. In this review, we discuss the knowledge gained on the biology and pathogenesis of M. leprae from current proteomic studies. © 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd.

GENOMIC AND PROTEOMIC TECHNIQUES APPLIED TO REPRODUCTIVE BIOLOGY

EPA Science Inventory

Genomic and proteomic techniques applied to reproductive biology
John C. Rockett
Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Research Tria...

Aptamer-Based Multiplexed Proteomic Technology for Biomarker Discovery

PubMed Central

Gold, Larry; Ayers, Deborah; Bertino, Jennifer; Bock, Christopher; Bock, Ashley; Brody, Edward N.; Carter, Jeff; Dalby, Andrew B.; Eaton, Bruce E.; Fitzwater, Tim; Flather, Dylan; Forbes, Ashley; Foreman, Trudi; Fowler, Cate; Gawande, Bharat; Goss, Meredith; Gunn, Magda; Gupta, Shashi; Halladay, Dennis; Heil, Jim; Heilig, Joe; Hicke, Brian; Husar, Gregory; Janjic, Nebojsa; Jarvis, Thale; Jennings, Susan; Katilius, Evaldas; Keeney, Tracy R.; Kim, Nancy; Koch, Tad H.; Kraemer, Stephan; Kroiss, Luke; Le, Ngan; Levine, Daniel; Lindsey, Wes; Lollo, Bridget; Mayfield, Wes; Mehan, Mike; Mehler, Robert; Nelson, Sally K.; Nelson, Michele; Nieuwlandt, Dan; Nikrad, Malti; Ochsner, Urs; Ostroff, Rachel M.; Otis, Matt; Parker, Thomas; Pietrasiewicz, Steve; Resnicow, Daniel I.; Rohloff, John; Sanders, Glenn; Sattin, Sarah; Schneider, Daniel; Singer, Britta; Stanton, Martin; Sterkel, Alana; Stewart, Alex; Stratford, Suzanne; Vaught, Jonathan D.; Vrkljan, Mike; Walker, Jeffrey J.; Watrobka, Mike; Waugh, Sheela; Weiss, Allison; Wilcox, Sheri K.; Wolfson, Alexey; Wolk, Steven K.; Zhang, Chi; Zichi, Dom

2010-01-01

Background The interrogation of proteomes (“proteomics”) in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. Methodology/Principal Findings We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (∼100 fM–1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states. Conclusions/Significance We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine. PMID:21165148

Omics for Precious Rare Biosamples: Characterization of Ancient Human Hair by a Proteomic Approach.

PubMed

Fresnais, Margaux; Richardin, Pascale; Sepúlveda, Marcela; Leize-Wagner, Emmanuelle; Charrié-Duhaut, Armelle

2017-07-01

Omics technologies have far-reaching applications beyond clinical medicine. A case in point is the analysis of ancient hair samples. Indeed, hair is an important biological indicator that has become a material of choice in archeometry to study the ancient civilizations and their environment. Current characterization of ancient hair is based on elemental and structural analyses, but only few studies have focused on the molecular aspects of ancient hair proteins-keratins-and their conservation state. In such cases, applied extraction protocols require large amounts of raw hair, from 30 to 100 mg. In the present study, we report an optimized new proteomic approach to accurately identify archeological hair proteins, and assess their preservation state, while using a minimum of raw material. Testing and adaptation of three protocols and of nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) parameters were performed on modern hair. On the basis of mass spectrometry data quality, and of the required initial sample amount, the most promising workflow was selected and applied to an ancient archeological sample, dated to about 3880 years before present. Finally, and importantly, we were able to identify 11 ancient hair proteins and to visualize the preservation state of mummy's hair from only 500 μg of raw material. The results presented here pave the way for new insights into the understanding of hair protein alteration processes such as those due to aging and ecological exposures. This work could enable omics scientists to apply a proteomic approach to precious and rare samples, not only in the context of archeometrical studies but also for future applications that would require the use of very small amounts of sample.

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.

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.

Quantitative proteomics in cardiovascular research: global and targeted strategies

PubMed Central

Shen, Xiaomeng; Young, Rebeccah; Canty, John M.; Qu, Jun

2014-01-01

Extensive technical advances in the past decade have substantially expanded quantitative proteomics in cardiovascular research. This has great promise for elucidating the mechanisms of cardiovascular diseases (CVD) and the discovery of cardiac biomarkers used for diagnosis and treatment evaluation. Global and targeted proteomics are the two major avenues of quantitative proteomics. While global approaches enable unbiased discovery of altered proteins via relative quantification at the proteome level, targeted techniques provide higher sensitivity and accuracy, and are capable of multiplexed absolute quantification in numerous clinical/biological samples. While promising, technical challenges need to be overcome to enable full utilization of these techniques in cardiovascular medicine. Here we discuss recent advances in quantitative proteomics and summarize applications in cardiovascular research with an emphasis on biomarker discovery and elucidating molecular mechanisms of disease. We propose the integration of global and targeted strategies as a high-throughput pipeline for cardiovascular proteomics. Targeted approaches enable rapid, extensive validation of biomarker candidates discovered by global proteomics. These approaches provide a promising alternative to immunoassays and other low-throughput means currently used for limited validation. PMID:24920501

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.

Proteogenomics: Integrating Next-Generation Sequencing and Mass Spectrometry to Characterize Human Proteomic Variation

NASA Astrophysics Data System (ADS)

Sheynkman, Gloria M.; Shortreed, Michael R.; Cesnik, Anthony J.; Smith, Lloyd M.

2016-06-01

Mass spectrometry-based proteomics has emerged as the leading method for detection, quantification, and characterization of proteins. Nearly all proteomic workflows rely on proteomic databases to identify peptides and proteins, but these databases typically contain a generic set of proteins that lack variations unique to a given sample, precluding their detection. Fortunately, proteogenomics enables the detection of such proteomic variations and can be defined, broadly, as the use of nucleotide sequences to generate candidate protein sequences for mass spectrometry database searching. Proteogenomics is experiencing heightened significance due to two developments: (a) advances in DNA sequencing technologies that have made complete sequencing of human genomes and transcriptomes routine, and (b) the unveiling of the tremendous complexity of the human proteome as expressed at the levels of genes, cells, tissues, individuals, and populations. We review here the field of human proteogenomics, with an emphasis on its history, current implementations, the types of proteomic variations it reveals, and several important applications.

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

AgHalo: A Facile Fluorogenic Sensor to Detect Drug-Induced Proteome Stress.

PubMed

Liu, Yu; Fares, Matthew; Dunham, Noah P; Gao, Zi; Miao, Kun; Jiang, Xueyuan; Bollinger, Samuel S; Boal, Amie K; Zhang, Xin

2017-07-17

Drug-induced proteome stress that involves protein aggregation may cause adverse effects and undermine the safety profile of a drug. Safety of drugs is regularly evaluated using cytotoxicity assays that measure cell death. However, these assays provide limited insights into the presence of proteome stress in live cells. A fluorogenic protein sensor is reported to detect drug-induced proteome stress prior to cell death. An aggregation prone Halo-tag mutant (AgHalo) was evolved to sense proteome stress through its aggregation. Detection of such conformational changes was enabled by a fluorogenic ligand that fluoresces upon AgHalo forming soluble aggregates. Using 5 common anticancer drugs, we exemplified detection of differential proteome stress before any cell death was observed. Thus, this sensor can be used to evaluate drug safety in a regime that the current cytotoxicity assays cannot cover and be generally applied to detect proteome stress induced by other toxins. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteogenomics: Integrating Next-Generation Sequencing and Mass Spectrometry to Characterize Human Proteomic Variation

PubMed Central

Sheynkman, Gloria M.; Shortreed, Michael R.; Cesnik, Anthony J.; Smith, Lloyd M.

2016-01-01

Mass spectrometry–based proteomics has emerged as the leading method for detection, quantification, and characterization of proteins. Nearly all proteomic workflows rely on proteomic databases to identify peptides and proteins, but these databases typically contain a generic set of proteins that lack variations unique to a given sample, precluding their detection. Fortunately, proteogenomics enables the detection of such proteomic variations and can be defined, broadly, as the use of nucleotide sequences to generate candidate protein sequences for mass spectrometry database searching. Proteogenomics is experiencing heightened significance due to two developments: (a) advances in DNA sequencing technologies that have made complete sequencing of human genomes and transcriptomes routine, and (b) the unveiling of the tremendous complexity of the human proteome as expressed at the levels of genes, cells, tissues, individuals, and populations. We review here the field of human proteogenomics, with an emphasis on its history, current implementations, the types of proteomic variations it reveals, and several important applications. PMID:27049631

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 ).

TrSDB: a proteome database of transcription factors

PubMed Central

Hermoso, Antoni; Aguilar, Daniel; Aviles, Francesc X.; Querol, Enrique

2004-01-01

TrSDB—TranScout Database—(http://ibb.uab.es/trsdb) is a proteome database of eukaryotic transcription factors based upon predicted motifs by TranScout and data sources such as InterPro and Gene Ontology Annotation. Nine eukaryotic proteomes are included in the current version. Extensive and diverse information for each database entry, different analyses considering TranScout classification and similarity relationships are offered for research on transcription factors or gene expression. PMID:14681387

Recognition of the polycistronic nature of human genes is critical to understanding the genotype-phenotype relationship.

PubMed

Brunet, Marie A; Levesque, Sébastien A; Hunting, Darel J; Cohen, Alan A; Roucou, Xavier

2018-05-01

Technological advances promise unprecedented opportunities for whole exome sequencing and proteomic analyses of populations. Currently, data from genome and exome sequencing or proteomic studies are searched against reference genome annotations. This provides the foundation for research and clinical screening for genetic causes of pathologies. However, current genome annotations substantially underestimate the proteomic information encoded within a gene. Numerous studies have now demonstrated the expression and function of alternative (mainly small, sometimes overlapping) ORFs within mature gene transcripts. This has important consequences for the correlation of phenotypes and genotypes. Most alternative ORFs are not yet annotated because of a lack of evidence, and this absence from databases precludes their detection by standard proteomic methods, such as mass spectrometry. Here, we demonstrate how current approaches tend to overlook alternative ORFs, hindering the discovery of new genetic drivers and fundamental research. We discuss available tools and techniques to improve identification of proteins from alternative ORFs and finally suggest a novel annotation system to permit a more complete representation of the transcriptomic and proteomic information contained within a gene. Given the crucial challenge of distinguishing functional ORFs from random ones, the suggested pipeline emphasizes both experimental data and conservation signatures. The addition of alternative ORFs in databases will render identification less serendipitous and advance the pace of research and genomic knowledge. This review highlights the urgent medical and research need to incorporate alternative ORFs in current genome annotations and thus permit their inclusion in hypotheses and models, which relate phenotypes and genotypes. © 2018 Brunet et al.; Published by Cold Spring Harbor Laboratory Press.

Proteome Studies of Filamentous Fungi

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

Baker, Scott E.; Panisko, Ellen A.

2011-04-20

The continued fast pace of fungal genome sequence generation has enabled proteomic analysis of a wide breadth of organisms that span the breadth of the Kingdom Fungi. There is some phylogenetic bias to the current catalog of fungi with reasonable DNA sequence databases (genomic or EST) that could be analyzed at a global proteomic level. However, the rapid development of next generation sequencing platforms has lowered the cost of genome sequencing such that in the near future, having a genome sequence will no longer be a time or cost bottleneck for downstream proteomic (and transcriptomic) analyses. High throughput, non-gel basedmore » proteomics offers a snapshot of proteins present in a given sample at a single point in time. There are a number of different variations on the general method and technologies for identifying peptides in a given sample. We present a method that can serve as a “baseline” for proteomic studies of fungi.« less

Recent 5-year Findings and Technological Advances in the Proteomic Study of HIV-associated Disorders.

PubMed

Zhang, Lijun; Jia, Xiaofang; Jin, Jun-O; Lu, Hongzhou; Tan, Zhimi

2017-04-01

Human immunodeficiency virus-1 (HIV-1) mainly relies on host factors to complete its life cycle. Hence, it is very important to identify HIV-regulated host proteins. Proteomics is an excellent technique for this purpose because of its high throughput and sensitivity. In this review, we summarized current technological advances in proteomics, including general isobaric tags for relative and absolute quantitation (iTRAQ) and stable isotope labeling by amino acids in cell culture (SILAC), as well as subcellular proteomics and investigation of posttranslational modifications. Furthermore, we reviewed the applications of proteomics in the discovery of HIV-related diseases and HIV infection mechanisms. Proteins identified by proteomic studies might offer new avenues for the diagnosis and treatment of HIV infection and the related diseases. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Proteome studies of filamentous fungi.

PubMed

Baker, Scott E; Panisko, Ellen A

2011-01-01

The continued fast pace of fungal genome sequence generation has enabled proteomic analysis of a wide variety of organisms that span the breadth of the Kingdom Fungi. There is some phylogenetic bias to the current catalog of fungi with reasonable DNA sequence databases (genomic or EST) that could be analyzed at a global proteomic level. However, the rapid development of next generation sequencing platforms has lowered the cost of genome sequencing such that in the near future, having a genome sequence will no longer be a time or cost bottleneck for downstream proteomic (and transcriptomic) analyses. High throughput, nongel-based proteomics offers a snapshot of proteins present in a given sample at a single point in time. There are a number of variations on the general methods and technologies for identifying peptides in a given sample. We present a method that can serve as a "baseline" for proteomic studies of fungi.

Trans-Proteomic Pipeline, a standardized data processing pipeline for large-scale reproducible proteomics informatics

PubMed Central

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

2015-01-01

Democratization of genomics technologies has enabled the rapid determination of genotypes. More recently the democratization of comprehensive proteomics technologies is enabling the determination of the cellular phenotype and the molecular events that define its dynamic state. Core proteomic technologies include mass spectrometry to define protein sequence, protein:protein interactions, and protein post-translational modifications. Key enabling technologies for proteomics are bioinformatic pipelines to identify, quantitate, and summarize these events. The Trans-Proteomics Pipeline (TPP) is a robust open-source standardized data processing pipeline for large-scale reproducible quantitative mass spectrometry proteomics. It supports all major operating systems and instrument vendors via open data formats. Here we provide a review of the overall proteomics workflow supported by the TPP, its major tools, and how it can be used in its various modes from desktop to cloud computing. We describe new features for the TPP, including data visualization functionality. We conclude by describing some common perils that affect the analysis of tandem mass spectrometry datasets, as well as some major upcoming features. PMID:25631240

Trans-Proteomic Pipeline, a standardized data processing pipeline for large-scale reproducible proteomics informatics.

PubMed

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

2015-08-01

Democratization of genomics technologies has enabled the rapid determination of genotypes. More recently the democratization of comprehensive proteomics technologies is enabling the determination of the cellular phenotype and the molecular events that define its dynamic state. Core proteomic technologies include MS to define protein sequence, protein:protein interactions, and protein PTMs. Key enabling technologies for proteomics are bioinformatic pipelines to identify, quantitate, and summarize these events. The Trans-Proteomics Pipeline (TPP) is a robust open-source standardized data processing pipeline for large-scale reproducible quantitative MS proteomics. It supports all major operating systems and instrument vendors via open data formats. Here, we provide a review of the overall proteomics workflow supported by the TPP, its major tools, and how it can be used in its various modes from desktop to cloud computing. We describe new features for the TPP, including data visualization functionality. We conclude by describing some common perils that affect the analysis of MS/MS datasets, as well as some major upcoming features. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomics reveals the effects of sustained weight loss on the human plasma proteome.

PubMed

Geyer, Philipp E; Wewer Albrechtsen, Nicolai J; Tyanova, Stefka; Grassl, Niklas; Iepsen, Eva W; Lundgren, Julie; Madsbad, Sten; Holst, Jens J; Torekov, Signe S; Mann, Matthias

2016-12-22

Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill-defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed by a year of weight maintenance. Using mass spectrometry-based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual-specific protein levels with wide-ranging effects of losing weight on the plasma proteome reflected in 93 significantly affected proteins. The adipocyte-secreted SERPINF1 and apolipoprotein APOF1 were most significantly regulated with fold changes of -16% and +37%, respectively (P < 10 -13 ), and the entire apolipoprotein family showed characteristic differential regulation. Clinical laboratory parameters are reflected in the plasma proteome, and eight plasma proteins correlated better with insulin resistance than the known marker adiponectin. Nearly all study participants benefited from weight loss regarding a ten-protein inflammation panel defined from the proteomics data. We conclude that plasma proteome profiling broadly evaluates and monitors intervention in metabolic diseases. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

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.

Embryology in the era of proteomics.

PubMed

Katz-Jaffe, Mandy G; McReynolds, Susanna

2013-03-15

Proteomic technologies have begun providing evidence that viable embryos possess unique protein profiles. Some of these potential protein biomarkers have been identified as extracellular and could be used in the development of a noninvasive quantitative method for embryo assessment. The field of assisted reproductive technologies would benefit from defining the human embryonic proteome and secretome, thereby expanding our current knowledge of embryonic cellular processes. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Chemical Proteomic Approaches Targeting Cancer Stem Cells: A Review of Current Literature.

PubMed

Jung, Hye Jin

2017-01-01

Cancer stem cells (CSCs) have been proposed as central drivers of tumor initiation, progression, recurrence, and therapeutic resistance. Therefore, identifying stem-like cells within cancers and understanding their properties is crucial for the development of effective anticancer therapies. Recently, chemical proteomics has become a powerful tool to efficiently determine protein networks responsible for CSC pathophysiology and comprehensively elucidate molecular mechanisms of drug action against CSCs. This review provides an overview of major methodologies utilized in chemical proteomic approaches. In addition, recent successful chemical proteomic applications targeting CSCs are highlighted. Future direction of potential CSC research by integrating chemical genomic and proteomic data obtained from a single biological sample of CSCs are also suggested in this review. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Proteomics data exchange and storage: the need for common standards and public repositories.

PubMed

Jiménez, Rafael C; Vizcaíno, Juan Antonio

2013-01-01

Both the existence of data standards and public databases or repositories have been key factors behind the development of the existing "omics" approaches. In this book chapter we first review the main existing mass spectrometry (MS)-based proteomics resources: PRIDE, PeptideAtlas, GPMDB, and Tranche. Second, we report on the current status of the different proteomics data standards developed by the Proteomics Standards Initiative (PSI): the formats mzML, mzIdentML, mzQuantML, TraML, and PSI-MI XML are then reviewed. Finally, we present an easy way to query and access MS proteomics data in the PRIDE database, as a representative of the existing repositories, using the workflow management system (WMS) tool Taverna. Two different publicly available workflows are explained and described.

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

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.

Pre-fractionation strategies to resolve pea (Pisum sativum) sub-proteomes

PubMed Central

Meisrimler, Claudia-Nicole; Menckhoff, Ljiljana; Kukavica, Biljana M.; Lüthje, Sabine

2015-01-01

Legumes are important crop plants and pea (Pisum sativum L.) has been investigated as a model with respect to several physiological aspects. The sequencing of the pea genome has not been completed. Therefore, proteomic approaches are currently limited. Nevertheless, the increasing numbers of available EST-databases as well as the high homology of the pea and medicago genome (Medicago truncatula Gaertner) allow the successful identification of proteins. Due to the un-sequenced pea genome, pre-fractionation approaches have been used in pea proteomic surveys in the past. Aside from a number of selective proteome studies on crude extracts and the chloroplast, few studies have targeted other components such as the pea secretome, an important sub-proteome of interest due to its role in abiotic and biotic stress processes. The secretome itself can be further divided into different sub-proteomes (plasma membrane, apoplast, cell wall proteins). Cell fractionation in combination with different gel-electrophoresis, chromatography methods and protein identification by mass spectrometry are important partners to gain insight into pea sub-proteomes, post-translational modifications and protein functions. Overall, pea proteomics needs to link numerous existing physiological and biochemical data to gain further insight into adaptation processes, which play important roles in field applications. Future developments and directions in pea proteomics are discussed. PMID:26539198

ProCon - PROteomics CONversion tool.

PubMed

Mayer, Gerhard; Stephan, Christian; Meyer, Helmut E; Kohl, Michael; Marcus, Katrin; Eisenacher, Martin

2015-11-03

With the growing amount of experimental data produced in proteomics experiments and the requirements/recommendations of journals in the proteomics field to publicly make available data described in papers, a need for long-term storage of proteomics data in public repositories arises. For such an upload one needs proteomics data in a standardized format. Therefore, it is desirable, that the proprietary vendor's software will integrate in the future such an export functionality using the standard formats for proteomics results defined by the HUPO-PSI group. Currently not all search engines and analysis tools support these standard formats. In the meantime there is a need to provide user-friendly free-to-use conversion tools that can convert the data into such standard formats in order to support wet-lab scientists in creating proteomics data files ready for upload into the public repositories. ProCon is such a conversion tool written in Java for conversion of proteomics identification data into standard formats mzIdentML and Pride XML. It allows the conversion of Sequest™/Comet .out files, of search results from the popular and often used ProteomeDiscoverer® 1.x (x=versions 1.1 to1.4) software and search results stored in the LIMS systems ProteinScape® 1.3 and 2.1 into mzIdentML and PRIDE XML. This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015. Published by Elsevier B.V.

Implementation of proteomics for cancer research: past, present, and future.

PubMed

Karimi, Parisa; Shahrokni, Armin; Ranjbar, Mohammad R Nezami

2014-01-01

Cancer is the leading cause of the death, accounts for about 13% of all annual deaths worldwide. Many different fields of science are collaborating together studying cancer to improve our knowledge of this lethal disease, and find better solutions for diagnosis and treatment. Proteomics is one of the most recent and rapidly growing areas in molecular biology that helps understanding cancer from an omics data analysis point of view. The human proteome project was officially initiated in 2008. Proteomics enables the scientists to interrogate a variety of biospecimens for their protein contents and measure the concentrations of these proteins. Current necessary equipment and technologies for cancer proteomics are mass spectrometry, protein microarrays, nanotechnology and bioinformatics. In this paper, we provide a brief review on proteomics and its application in cancer research. After a brief introduction including its definition, we summarize the history of major previous work conducted by researchers, followed by an overview on the role of proteomics in cancer studies. We also provide a list of different utilities in cancer proteomics and investigate their advantages and shortcomings from theoretical and practical angles. Finally, we explore some of the main challenges and conclude the paper with future directions in this field.

Chemical composition and the potential for proteomic transformation in cancer, hypoxia, and hyperosmotic stress

PubMed Central

2017-01-01

The changes of protein expression that are monitored in proteomic experiments are a type of biological transformation that also involves changes in chemical composition. Accompanying the myriad molecular-level interactions that underlie any proteomic transformation, there is an overall thermodynamic potential that is sensitive to microenvironmental conditions, including local oxidation and hydration potential. Here, up- and down-expressed proteins identified in 71 comparative proteomics studies were analyzed using the average oxidation state of carbon (ZC) and water demand per residue (\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${\\overline{n}}_{{\\mathrm{H}}_{2}\\mathrm{O}}$\\end{document}n¯H2O), calculated using elemental abundances and stoichiometric reactions to form proteins from basis species. Experimental lowering of oxygen availability (hypoxia) or water activity (hyperosmotic stress) generally results in decreased ZC or \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${\\overline{n}}_{{\\mathrm{H}}_{2}\\mathrm{O}}$\\end{document}n¯H2O of up-expressed compared to down-expressed proteins. This correspondence of chemical composition with experimental conditions provides evidence for attraction of the proteomes to a low-energy state. An opposite compositional change, toward higher average oxidation or hydration state, is found for proteomic transformations in colorectal and pancreatic cancer, and in two experiments for adipose-derived stem cells. Calculations of chemical affinity were used to estimate the thermodynamic potentials for proteomic transformations as a function of fugacity of O2 and activity of H2O, which serve as scales of oxidation and hydration potential. Diagrams summarizing the relative potential for formation of up- and down-expressed proteins have predicted equipotential lines that cluster around particular values of oxygen fugacity and water activity for similar datasets. The changes in chemical composition of proteomes are likely linked with reactions among other cellular molecules. A redox balance calculation indicates that an increase in the lipid to protein ratio in cancer cells by 20% over hypoxic cells would generate a large enough electron sink for oxidation of the cancer proteomes. The datasets and computer code used here are made available in a new R package, canprot. PMID:28603672

CPTC and NIST-sponsored Yeast Reference Material Now Publicly Available | Office of Cancer Clinical Proteomics Research

Cancer.gov

The yeast protein extract (RM8323) developed by National Institute of Standards and Technology (NIST) under the auspices of NCI's CPTC initiative is currently available to the public at https://www-s.nist.gov/srmors/view_detail.cfm?srm=8323. The yeast proteome offers researchers a unique biological reference material. RM8323 is the most extensively characterized complex biological proteome and the only one associated with several large-scale studies to estimate protein abundance across a wide concentration range.

Scientific Workflow Management in Proteomics

PubMed Central

de Bruin, Jeroen S.; Deelder, André M.; Palmblad, Magnus

2012-01-01

Data processing in proteomics can be a challenging endeavor, requiring extensive knowledge of many different software packages, all with different algorithms, data format requirements, and user interfaces. In this article we describe the integration of a number of existing programs and tools in Taverna Workbench, a scientific workflow manager currently being developed in the bioinformatics community. We demonstrate how a workflow manager provides a single, visually clear and intuitive interface to complex data analysis tasks in proteomics, from raw mass spectrometry data to protein identifications and beyond. PMID:22411703

Proteome and phosphoproteome analysis of commensally induced dendritic cell maturation states.

PubMed

Korkmaz, Ali Giray; Popov, Todor; Peisl, Loulou; Codrea, Marius Cosmin; Nahnsen, Sven; Steimle, Alexander; Velic, Ana; Macek, Boris; von Bergen, Martin; Bernhardt, Joerg; Frick, Julia-Stefanie

2018-05-30

Dendritic cells (DCs) can shape the immune system towards an inflammatory or tolerant state depending on the bacterial antigens and the environment they encounter. In this study we provide a proteomic catalogue of differentially expressed proteins between distinct DC maturation states, brought about by bacteria that differ in their endotoxicity. To achieve this, we have performed proteomics and phosphoproteomics on murine DC cultures. Symbiont and pathobiont bacteria were used to direct dendritic cells into a semi-mature and fully-mature state, respectively. The comparison of semi-mature and fully-mature DCs revealed differential expression in 103 proteins and differential phosphorylation in 118 phosphosites, including major regulatory factors of central immune processes. Our analyses predict that these differences are mediated by upstream elements such as SOCS1, IRF3, ABCA1, TLR4, and PTGER4. Our analyses indicate that the symbiont bacterial strain affects DC proteome in a distinct way, by downregulating inflammatory proteins and activating anti-inflammatory upstream regulators. Biological significance In this study we have investigated the responses of immune cells to distinct bacterial stimuli. We have used the symbiont bacterial strain B. vulgatus and the pathobiont E. coli strain to stimulate cultured primary dendritic cells and performed a shotgun proteome analysis to investigate the protein expression and phosphorylation level differences on a genome level. We have observed expression and phosphorylation level differences in key immune regulators, transcription factors and signal transducers. Moreover, our subsequent bioinformatics analysis indicated regulation at several signaling pathways such as PPAR signaling, LXR/RXR activation and glucocorticoid signaling pathways, which are not studied in detail in an inflammation and DC maturation context. Our phosphoproteome analysis showed differential phosphorylation in 118 phosphosites including those belonging to epigenetic regulators, transcription factors and major cell cycle regulators. We anticipate that our study will facilitate further investigation of immune cell proteomes under different inflammatory and non-inflammatory conditions. Copyright © 2017. Published by Elsevier B.V.

Advances in Proteomics Data Analysis and Display Using an Accurate Mass and Time Tag Approach

PubMed Central

Zimmer, Jennifer S.D.; Monroe, Matthew E.; Qian, Wei-Jun; Smith, Richard D.

2007-01-01

Proteomics has recently demonstrated utility in understanding cellular processes on the molecular level as a component of systems biology approaches and for identifying potential biomarkers of various disease states. The large amount of data generated by utilizing high efficiency (e.g., chromatographic) separations coupled to high mass accuracy mass spectrometry for high-throughput proteomics analyses presents challenges related to data processing, analysis, and display. This review focuses on recent advances in nanoLC-FTICR-MS-based proteomics approaches and the accompanying data processing tools that have been developed to display and interpret the large volumes of data being produced. PMID:16429408

Tetrazine ligation for chemical proteomics.

PubMed

Kang, Kyungtae; Park, Jongmin; Kim, Eunha

2016-01-01

Determining small molecule-target protein interaction is essential for the chemical proteomics. One of the most important keys to explore biological system in chemical proteomics field is finding first-class molecular tools. Chemical probes can provide great spatiotemporal control to elucidate biological functions of proteins as well as for interrogating biological pathways. The invention of bioorthogonal chemistry has revolutionized the field of chemical biology by providing superior chemical tools and has been widely used for investigating the dynamics and function of biomolecules in live condition. Among 20 different bioorthogonal reactions, tetrazine ligation has been spotlighted as the most advanced bioorthogonal chemistry because of their extremely faster kinetics and higher specificity than others. Therefore, tetrazine ligation has a tremendous potential to enhance the proteomic research. This review highlights the current status of tetrazine ligation reaction as a molecular tool for the chemical proteomics.

Proteomics in the genome engineering era.

PubMed

Vandemoortele, Giel; Gevaert, Kris; Eyckerman, Sven

2016-01-01

Genome engineering experiments used to be lengthy, inefficient, and often expensive, preventing a widespread adoption of such experiments for the full assessment of endogenous protein functions. With the revolutionary clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 technology, genome engineering became accessible to the broad life sciences community and is now implemented in several research areas. One particular field that can benefit significantly from this evolution is proteomics where a substantial impact on experimental design and general proteome biology can be expected. In this review, we describe the main applications of genome engineering in proteomics, including the use of engineered disease models and endogenous epitope tagging. In addition, we provide an overview on current literature and highlight important considerations when launching genome engineering technologies in proteomics workflows. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos

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

Li, Zhou; Adams, Rachel M; Chourey, Karuna

2012-01-01

A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification. Isobaricmore » chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. Based on the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.« less

Application of proteomics in research on traditional Chinese medicine.

PubMed

Suo, Tongchuan; Wang, Haixia; Li, Zheng

2016-09-01

Traditional Chinese medicine (TCM) is a widely used complementary alternative medicine approach. Although many aspects of its effectiveness have been approved clinically, rigorous scientific techniques are highly required to translate the promises from TCM into powerful modern therapies. In this respect, proteomics is useful because of its ability to unveil the underlying target proteins and/or protein biomarkers. In this review, we summarize the recent interplay between proteomics and research on TCM, ranging from exploration of the medicinal materials to the biological basis of TCM concepts, and from pathological studies to pharmacological investigations. We show that proteomic analyses provide preliminary biological evidence of the promises in TCM, and the integration of proteomics with other omics and bioinformatics offers a comprehensive methodology to address the complications of TCM. Expert commentary: Currently, only limited information can be obtained regarding TCM issues and thus more work is required to resolve the ambiguity. As such, more collaborations between proteomics and other techniques (other omics, network pharmacology, etc.) are essential for deciphering the underlying biological basis in TCM topics.

Comparative and Quantitative Global Proteomics Approaches: An Overview

PubMed Central

Deracinois, Barbara; Flahaut, Christophe; Duban-Deweer, Sophie; Karamanos, Yannis

2013-01-01

Proteomics became a key tool for the study of biological systems. The comparison between two different physiological states allows unravelling the cellular and molecular mechanisms involved in a biological process. Proteomics can confirm the presence of proteins suggested by their mRNA content and provides a direct measure of the quantity present in a cell. Global and targeted proteomics strategies can be applied. Targeted proteomics strategies limit the number of features that will be monitored and then optimise the methods to obtain the highest sensitivity and throughput for a huge amount of samples. The advantage of global proteomics strategies is that no hypothesis is required, other than a measurable difference in one or more protein species between the samples. Global proteomics methods attempt to separate quantify and identify all the proteins from a given sample. This review highlights only the different techniques of separation and quantification of proteins and peptides, in view of a comparative and quantitative global proteomics analysis. The in-gel and off-gel quantification of proteins will be discussed as well as the corresponding mass spectrometry technology. The overview is focused on the widespread techniques while keeping in mind that each approach is modular and often recovers the other. PMID:28250403

The porcine translational research database: A manually curated, genomics and proteomics-based research resource

USDA-ARS?s Scientific Manuscript database

The use of swine in biomedical research has increased dramatically in the last decade. Diverse genomic- and proteomic databases have been developed to facilitate research using human and rodent models. Current porcine gene databases, however, lack the robust annotation to study pig models that are...

Proteomics and Systems Biology: Current and Future Applications in the Nutritional Sciences1

PubMed Central

Moore, J. Bernadette; Weeks, Mark E.

2011-01-01

In the last decade, advances in genomics, proteomics, and metabolomics have yielded large-scale datasets that have driven an interest in global analyses, with the objective of understanding biological systems as a whole. Systems biology integrates computational modeling and experimental biology to predict and characterize the dynamic properties of biological systems, which are viewed as complex signaling networks. Whereas the systems analysis of disease-perturbed networks holds promise for identification of drug targets for therapy, equally the identified critical network nodes may be targeted through nutritional intervention in either a preventative or therapeutic fashion. As such, in the context of the nutritional sciences, it is envisioned that systems analysis of normal and nutrient-perturbed signaling networks in combination with knowledge of underlying genetic polymorphisms will lead to a future in which the health of individuals will be improved through predictive and preventative nutrition. Although high-throughput transcriptomic microarray data were initially most readily available and amenable to systems analysis, recent technological and methodological advances in MS have contributed to a linear increase in proteomic investigations. It is now commonplace for combined proteomic technologies to generate complex, multi-faceted datasets, and these will be the keystone of future systems biology research. This review will define systems biology, outline current proteomic methodologies, highlight successful applications of proteomics in nutrition research, and discuss the challenges for future applications of systems biology approaches in the nutritional sciences. PMID:22332076

Capillary electrophoresis interfaced with a mass spectrometer (CE-MS): technical considerations and applicability for biomarker studies in animals.

PubMed

Albalat, Amaya; Husi, Holger; Siwy, Justyna; Nally, Jarlath E; McLauglin, Mark; Eckersall, Peter D; Mullen, William

2014-02-01

Proteomics is a growing field that has the potential to be applied to many biology-related disciplines. However, the study of the proteome has proven to be very challenging due to its high level of complexity when compared to genome and transcriptome data. In order to analyse this level of complexity, high resolution separation of peptides/proteins are needed together with high resolution analysers. Currently, liquid chromatography and capillary electrophoresis (CE) are the two most widely used separation techniques that can be coupled on-line with a mass spectrometer (MS). In CE, proteins/ peptides are separated according to their size, charge and shape leading to high resolving power. Although further progress in the area of sensitivity, throughput and proteome coverage are expected, MS-based proteomics have developed to a level at which they are habitually applied to study a wide range of biological questions. The aim of this review is to present CE-MS as a proteomic analytical platform for biomarker research that could be used in farm animal and veterinary studies. This is a MS-analytical platform that has been widely used for biomarker research in the biomedical field but its application in animal proteomic studies is relatively novel. The review will focus on introducing the CE-MS platform and the primary considerations for its application to biomarker research. Furthermore, current applications but more importantly potential application in the field of farm animals and veterinary science will be presented and discussed.

A fresh look at the male-specific region of the human Y chromosome.

PubMed

Jangravi, Zohreh; Alikhani, Mehdi; Arefnezhad, Babak; Sharifi Tabar, Mehdi; Taleahmad, Sara; Karamzadeh, Razieh; Jadaliha, Mahdieh; Mousavi, Seyed Ahmad; Ahmadi Rastegar, Diba; Parsamatin, Pouria; Vakilian, Haghighat; Mirshahvaladi, Shahab; Sabbaghian, Marjan; Mohseni Meybodi, Anahita; Mirzaei, Mehdi; Shahhoseini, Maryam; Ebrahimi, Marzieh; Piryaei, Abbas; Moosavi-Movahedi, Ali Akbar; Haynes, Paul A; Goodchild, Ann K; Nasr-Esfahani, Mohammad Hossein; Jabbari, Esmaiel; Baharvand, Hossein; Sedighi Gilani, Mohammad Ali; Gourabi, Hamid; Salekdeh, Ghasem Hosseini

2013-01-04

The Chromosome-centric Human Proteome Project (C-HPP) aims to systematically map the entire human proteome with the intent to enhance our understanding of human biology at the cellular level. This project attempts simultaneously to establish a sound basis for the development of diagnostic, prognostic, therapeutic, and preventive medical applications. In Iran, current efforts focus on mapping the proteome of the human Y chromosome. The male-specific region of the Y chromosome (MSY) is unique in many aspects and comprises 95% of the chromosome's length. The MSY continually retains its haploid state and is full of repeated sequences. It is responsible for important biological roles such as sex determination and male fertility. Here, we present the most recent update of MSY protein-encoding genes and their association with various traits and diseases including sex determination and reversal, spermatogenesis and male infertility, cancers such as prostate cancers, sex-specific effects on the brain and behavior, and graft-versus-host disease. We also present information available from RNA sequencing, protein-protein interaction, post-translational modification of MSY protein-coding genes and their implications in biological systems. An overview of Human Y chromosome Proteome Project is presented and a systematic approach is suggested to ensure that at least one of each predicted protein-coding gene's major representative proteins will be characterized in the context of its major anatomical sites of expression, its abundance, and its functional relevance in a biological and/or medical context. There are many technical and biological issues that will need to be overcome in order to accomplish the full scale mapping.

The need for agriculture phenotyping: "moving from genotype to phenotype".

PubMed

Boggess, Mark V; Lippolis, John D; Hurkman, William J; Fagerquist, Clifton K; Briggs, Steve P; Gomes, Aldrin V; Righetti, Pier Giorgio; Bala, Kumar

2013-11-20

Increase in the world population has called for the increased demand for agricultural productivity. Traditional methods to augment crop and animal production are facing exacerbating pressures in keeping up with population growth. This challenge has in turn led to the transformational change in the use of biotechnology tools to meet increased productivity for both plant and animal systems. Although many challenges exist, the use of proteomic techniques to understand agricultural problems is steadily increasing. This review discusses the impact of genomics, proteomics, metabolomics and phenotypes on plant, animal and bacterial systems to achieve global food security and safety and we highlight examples of intra and extra mural research work that is currently being done to increase agricultural productivity. This review focuses on the global demand for increased agricultural productivity arising from population growth and how we can address this challenge using biotechnology. With a population well above seven billion humans, in a very unbalanced nutritional state (20% overweight, 20% risking starvation) drastic measures have to be taken at the political, infrastructure and scientific levels. While we cannot influence politics, it is our duty as scientists to see what can be done to feed humanity. Hence we highlight the transformational change in the use of biotechnology tools over traditional methods to increase agricultural productivity (plant and animal). Specifically, this review deals at length on how a three-pronged attack, namely combined genomics, proteomics and metabolomics, can help to ensure global food security and safety. This article is part of a Special Issue entitled: Translational Plant Proteomics. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

PubMed Central

Castagnola, M.; Scarano, E.; Messana, I.; Cabras, T.; Iavarone, F.; Di Cintio, G.; Fiorita, A.; De Corso, E.; Paludetti, G.

2017-01-01

SUMMARY Saliva testing is a non-invasive and inexpensive test that can serve as a source of information useful for diagnosis of disease. As we enter the era of genomic technologies and –omic research, collection of saliva has increased. Recent proteomic platforms have analysed the human salivary proteome and characterised about 3000 differentially expressed proteins and peptides: in saliva, more than 90% of proteins in weight are derived from the secretion of three couples of "major" glands; all the other components are derived from minor glands, gingival crevicular fluid, mucosal exudates and oral microflora. The most common aim of proteomic analysis is to discriminate between physiological and pathological conditions. A proteomic protocol to analyze the whole saliva proteome is not currently available. It is possible distinguish two type of proteomic platforms: top-down proteomics investigates intact naturally-occurring structure of a protein under examination; bottom-up proteomics analyses peptide fragments after pre-digestion (typically with trypsin). Because of this heterogeneity, many different biomarkers may be proposed for the same pathology. The salivary proteome has been characterised in several diseases: oral squamous cell carcinoma and oral leukoplakia, chronic graft-versus-host disease Sjögren's syndrome and other autoimmune disorders such as SAPHO, schizophrenia and bipolar disorder, and genetic diseases like Down's Syndrome and Wilson disease. The results of research reported herein suggest that in the near future human saliva will be a relevant diagnostic fluid for clinical diagnosis and prognosis. PMID:28516971

ICPC Pilots International Student Training, Paving a Path for Tomorrow’s Cancer Researchers | Office of Cancer Clinical Proteomics Research

Cancer.gov

The Office of Cancer Clinical Proteomics Research at the National Cancer Institute, part of the United States National Institutes of Health, is spearheading the preparationand training of the proteogenomic research workforce on an international scale.

ADVANCED PROTEOMICS AND BIOINFORMATICS TOOLS IN TOXICOLOGY RESEARCH: OVERCOMING CHALLENGES TO PROVIDE SIGNIFICANT RESULTS

EPA Science Inventory

This presentation specifically addresses the advantages and limitations of state of the art gel, protein arrays and peptide-based labeling proteomic approaches to assess the effects of a suite of model T4 inhibitors on the thyroid axis of Xenopus laevis.

Current trends in mass spectrometry of peptides and proteins: Application to veterinary and sports-doping control.

PubMed

van den Broek, Irene; Blokland, Marco; Nessen, Merel A; Sterk, Saskia

2015-01-01

Detection of misuse of peptides and proteins as growth promoters is a major issue for sport and food regulatory agencies. The limitations of current analytical detection strategies for this class of compounds, in combination with their efficacy in growth-promoting effects, make peptide and protein drugs highly susceptible to abuse by either athletes or farmers who seek for products to illicitly enhance muscle growth. Mass spectrometry (MS) for qualitative analysis of peptides and proteins is well-established, particularly due to tremendous efforts in the proteomics community. Similarly, due to advancements in targeted proteomic strategies and the rapid growth of protein-based biopharmaceuticals, MS for quantitative analysis of peptides and proteins is becoming more widely accepted. These continuous advances in MS instrumentation and MS-based methodologies offer enormous opportunities for detection and confirmation of peptides and proteins. Therefore, MS seems to be the method of choice to improve the qualitative and quantitative analysis of peptide and proteins with growth-promoting properties. This review aims to address the opportunities of MS for peptide and protein analysis in veterinary control and sports-doping control with a particular focus on detection of illicit growth promotion. An overview of potential peptide and protein targets, including their amino acid sequence characteristics and current MS-based detection strategies is, therefore, provided. Furthermore, improvements of current and new detection strategies with state-of-the-art MS instrumentation are discussed for qualitative and quantitative approaches. © 2013 Wiley Periodicals, Inc.

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.

A Method for Label-Free, Differential Top-Down Proteomics.

PubMed

Ntai, Ioanna; Toby, Timothy K; LeDuc, Richard D; Kelleher, Neil L

2016-01-01

Biomarker discovery in the translational research has heavily relied on labeled and label-free quantitative bottom-up proteomics. Here, we describe a new approach to biomarker studies that utilizes high-throughput top-down proteomics and is the first to offer whole protein characterization and relative quantitation within the same experiment. Using yeast as a model, we report procedures for a label-free approach to quantify the relative abundance of intact proteins ranging from 0 to 30 kDa in two different states. In this chapter, we describe the integrated methodology for the large-scale profiling and quantitation of the intact proteome by liquid chromatography-mass spectrometry (LC-MS) without the need for metabolic or chemical labeling. This recent advance for quantitative top-down proteomics is best implemented with a robust and highly controlled sample preparation workflow before data acquisition on a high-resolution mass spectrometer, and the application of a hierarchical linear statistical model to account for the multiple levels of variance contained in quantitative proteomic comparisons of samples for basic and clinical research.

Methodologies and Perspectives of Proteomics Applied to Filamentous Fungi: From Sample Preparation to Secretome Analysis

PubMed Central

Bianco, Linda; Perrotta, Gaetano

2015-01-01

Filamentous fungi possess the extraordinary ability to digest complex biomasses and mineralize numerous xenobiotics, as consequence of their aptitude to sensing the environment and regulating their intra and extra cellular proteins, producing drastic changes in proteome and secretome composition. Recent advancement in proteomic technologies offers an exciting opportunity to reveal the fluctuations of fungal proteins and enzymes, responsible for their metabolic adaptation to a large variety of environmental conditions. Here, an overview of the most commonly used proteomic strategies will be provided; this paper will range from sample preparation to gel-free and gel-based proteomics, discussing pros and cons of each mentioned state-of-the-art technique. The main focus will be kept on filamentous fungi. Due to the biotechnological relevance of lignocellulose degrading fungi, special attention will be finally given to their extracellular proteome, or secretome. Secreted proteins and enzymes will be discussed in relation to their involvement in bio-based processes, such as biomass deconstruction and mycoremediation. PMID:25775160

Methodologies and perspectives of proteomics applied to filamentous fungi: from sample preparation to secretome analysis.

PubMed

Bianco, Linda; Perrotta, Gaetano

2015-03-12

Filamentous fungi possess the extraordinary ability to digest complex biomasses and mineralize numerous xenobiotics, as consequence of their aptitude to sensing the environment and regulating their intra and extra cellular proteins, producing drastic changes in proteome and secretome composition. Recent advancement in proteomic technologies offers an exciting opportunity to reveal the fluctuations of fungal proteins and enzymes, responsible for their metabolic adaptation to a large variety of environmental conditions. Here, an overview of the most commonly used proteomic strategies will be provided; this paper will range from sample preparation to gel-free and gel-based proteomics, discussing pros and cons of each mentioned state-of-the-art technique. The main focus will be kept on filamentous fungi. Due to the biotechnological relevance of lignocellulose degrading fungi, special attention will be finally given to their extracellular proteome, or secretome. Secreted proteins and enzymes will be discussed in relation to their involvement in bio-based processes, such as biomass deconstruction and mycoremediation.

Exploring the post-genomic world: differing explanatory and manipulatory functions of post-genomic sciences

PubMed Central

Holmes, Christina; Carlson, Siobhan M.; McDonald, Fiona; Jones, Mavis; Graham, Janice

2016-01-01

Richard Lewontin proposed that the ability of a scientific field to create a narrative for public understanding garners it social relevance. This article applies Lewontin's conceptual framework of the functions of science (manipulatory and explanatory) to compare and explain the current differences in perceived societal relevance of genetics/genomics and proteomics. We provide three examples to illustrate the social relevance and strong cultural narrative of genetics/genomics for which no counterpart exists for proteomics. We argue that the major difference between genetics/genomics and proteomics is that genomics has a strong explanatory function, due to the strong cultural narrative of heredity. Based on qualitative interviews and observations of proteomics conferences, we suggest that the nature of proteins, lack of public understanding, and theoretical complexity exacerbates this difference for proteomics. Lewontin's framework suggests that social scientists may find that omics sciences affect social relations in different ways than past analyses of genetics. PMID:27134568

Exploring the post-genomic world: differing explanatory and manipulatory functions of post-genomic sciences.

PubMed

Holmes, Christina; Carlson, Siobhan M; McDonald, Fiona; Jones, Mavis; Graham, Janice

2016-01-02

Richard Lewontin proposed that the ability of a scientific field to create a narrative for public understanding garners it social relevance. This article applies Lewontin's conceptual framework of the functions of science (manipulatory and explanatory) to compare and explain the current differences in perceived societal relevance of genetics/genomics and proteomics. We provide three examples to illustrate the social relevance and strong cultural narrative of genetics/genomics for which no counterpart exists for proteomics. We argue that the major difference between genetics/genomics and proteomics is that genomics has a strong explanatory function, due to the strong cultural narrative of heredity. Based on qualitative interviews and observations of proteomics conferences, we suggest that the nature of proteins, lack of public understanding, and theoretical complexity exacerbates this difference for proteomics. Lewontin's framework suggests that social scientists may find that omics sciences affect social relations in different ways than past analyses of genetics.

Proteomics of blood-based therapeutics: a promising tool for quality assurance in transfusion medicine.

PubMed

Thiele, Thomas; Steil, Leif; Völker, Uwe; Greinacher, Andreas

2007-01-01

Blood-based therapeutics are cellular or plasma components derived from human blood. Their production requires appropriate selection and treatment of the donor and processing of cells or plasma proteins. In contrast to clearly defined, chemically synthesized drugs, blood-derived therapeutics are highly complex mixtures of plasma proteins or even more complex cells. Pathogen transmission by the product as well as changes in the integrity of blood constituents resulting in loss of function or immune modulation are currently important issues in transfusion medicine. Protein modifications can occur during various steps of the production process, such as acquisition, enrichment of separate components (e.g. coagulation factors, cell populations), virus inactivation, conservation, and storage. Contemporary proteomic strategies allow a comprehensive assessment of protein modifications with high coverage, offer capabilities for qualitative and even quantitative analysis, and for high-throughput protein identification. Traditionally, proteomics approaches predominantly relied on two-dimensional gel electrophoresis (2-DE). Even if 2-DE is still state of the art, it has inherent limitations that are mainly based on the physicochemical properties of the proteins analyzed; for example, proteins with extremes in molecular mass and hydrophobicity (most membrane proteins) are difficult to assess by 2-DE. These limitations have fostered the development of mass spectrometry centered on non-gel-based separation approaches, which have proven to be highly successful and are thus complementing and even partially replacing 2-DE-based approaches. Although blood constituents have been extensively analyzed by proteomics, this technology has not been widely applied to assess or even improve blood-derived therapeutics, or to monitor the production processes. As proteomic technologies have the capacity to provide comprehensive information about changes occurring during processing and storage of blood products, proteomics can potentially guide improvement of pathogen inactivation procedures and engineering of stem cells, and may also allow a better understanding of factors influencing the immunogenicity of blood-derived therapeutics. An important development in proteomics is the reduction of inter-assay variability. This now allows the screening of samples taken from the same product over time or before and after processing. Optimized preparation procedures and storage conditions will reduce the risk of protein alterations, which in turn may contribute to better recovery, reduced exposure to allogeneic proteins, and increased transfusion safety.

Technological advances in real-time tracking of cell death

PubMed Central

Skommer, Joanna; Darzynkiewicz, Zbigniew; Wlodkowic, Donald

2010-01-01

Cell population can be viewed as a quantum system, which like Schrödinger’s cat exists as a combination of survival- and death-allowing states. Tracking and understanding cell-to-cell variability in processes of high spatio-temporal complexity such as cell death is at the core of current systems biology approaches. As probabilistic modeling tools attempt to impute information inaccessible by current experimental approaches, advances in technologies for single-cell imaging and omics (proteomics, genomics, metabolomics) should go hand in hand with the computational efforts. Over the last few years we have made exciting technological advances that allow studies of cell death dynamically in real-time and with the unprecedented accuracy. These approaches are based on innovative fluorescent assays and recombinant proteins, bioelectrical properties of cells, and more recently also on state-of-the-art optical spectroscopy. Here, we review current status of the most innovative analytical technologies for dynamic tracking of cell death, and address the interdisciplinary promises and future challenges of these methods. PMID:20519963

Proteomic and oxi-proteomic response of apple to a compatible (p. expansum) and a non-host (p. digitatum) pathogen

USDA-ARS?s Scientific Manuscript database

Despite the current use of chemical fungicides, Penicillium expansum still is one of the most devastating pathogens of pome fruit. In particular, P. expansum enters tissues through wounds causing large economic losses worldwide. To obtain new rational and environmental friendly control alternative...

Clinical proteomic biomarkers: relevant issues on study design & technical considerations in biomarker development

PubMed Central

2014-01-01

Biomarker research is continuously expanding in the field of clinical proteomics. A combination of different proteomic–based methodologies can be applied depending on the specific clinical context of use. Moreover, current advancements in proteomic analytical platforms are leading to an expansion of biomarker candidates that can be identified. Specifically, mass spectrometric techniques could provide highly valuable tools for biomarker research. Ideally, these advances could provide with biomarkers that are clinically applicable for disease diagnosis and/ or prognosis. Unfortunately, in general the biomarker candidates fail to be implemented in clinical decision making. To improve on this current situation, a well-defined study design has to be established driven by a clear clinical need, while several checkpoints between the different phases of discovery, verification and validation have to be passed in order to increase the probability of establishing valid biomarkers. In this review, we summarize the technical proteomic platforms that are available along the different stages in the biomarker discovery pipeline, exemplified by clinical applications in the field of bladder cancer biomarker research. PMID:24679154

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.

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.

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

Making a protein extract from plant pathogenic fungi for gel- and LC-based proteomics.

PubMed

Fernández, Raquel González; Redondo, Inmaculada; Jorrin-Novo, Jesus V

2014-01-01

Proteomic technologies have become a successful tool to provide relevant information on fungal biology. In the case of plant pathogenic fungi, this approach would allow a deeper knowledge of the interaction and the biological cycle of the pathogen, as well as the identification of pathogenicity and virulence factors. These two elements open up new possibilities for crop disease diagnosis and environment-friendly crop protection. Phytopathogenic fungi, due to its particular cellular characteristics, can be considered as a recalcitrant biological material, which makes it difficult to obtain quality protein samples for proteomic analysis. This chapter focuses on protein extraction for gel- and LC-based proteomics with specific protocols of our current research with Botrytis cinerea.

Comparative Analysis of Predicted Plastid-Targeted Proteomes of Sequenced Higher Plant Genomes

PubMed Central

Schaeffer, Scott; Harper, Artemus; Raja, Rajani; Jaiswal, Pankaj; Dhingra, Amit

2014-01-01

Plastids are actively involved in numerous plant processes critical to growth, development and adaptation. They play a primary role in photosynthesis, pigment and monoterpene synthesis, gravity sensing, starch and fatty acid synthesis, as well as oil, and protein storage. We applied two complementary methods to analyze the recently published apple genome (Malus × domestica) to identify putative plastid-targeted proteins, the first using TargetP and the second using a custom workflow utilizing a set of predictive programs. Apple shares roughly 40% of its 10,492 putative plastid-targeted proteins with that of the Arabidopsis (Arabidopsis thaliana) plastid-targeted proteome as identified by the Chloroplast 2010 project and ∼57% of its entire proteome with Arabidopsis. This suggests that the plastid-targeted proteomes between apple and Arabidopsis are different, and interestingly alludes to the presence of differential targeting of homologs between the two species. Co-expression analysis of 2,224 genes encoding putative plastid-targeted apple proteins suggests that they play a role in plant developmental and intermediary metabolism. Further, an inter-specific comparison of Arabidopsis, Prunus persica (Peach), Malus × domestica (Apple), Populus trichocarpa (Black cottonwood), Fragaria vesca (Woodland Strawberry), Solanum lycopersicum (Tomato) and Vitis vinifera (Grapevine) also identified a large number of novel species-specific plastid-targeted proteins. This analysis also revealed the presence of alternatively targeted homologs across species. Two separate analyses revealed that a small subset of proteins, one representing 289 protein clusters and the other 737 unique protein sequences, are conserved between seven plastid-targeted angiosperm proteomes. Majority of the novel proteins were annotated to play roles in stress response, transport, catabolic processes, and cellular component organization. Our results suggest that the current state of knowledge regarding plastid biology, preferentially based on model systems is deficient. New plant genomes are expected to enable the identification of potentially new plastid-targeted proteins that will aid in studying novel roles of plastids. PMID:25393533

Surviving the infarct: A profile of cardiac myosin binding protein-C pathogenicity, diagnostic utility, and proteomics in the ischemic myocardium.

PubMed

Lynch, Thomas L; Sadayappan, Sakthivel

2014-08-01

Cardiac myosin binding protein-C (cMyBP-C) is a regulatory protein of the contractile apparatus within the cardiac sarcomere. Ischemic injury to the heart during myocardial infarction (MI) results in the cleavage of cMyBP-C in a phosphorylation-dependent manner and release of an N-terminal fragment (C0C1f) into the circulation. C0C1f has been shown to be pathogenic within cardiac tissue, leading to the development of heart failure. Based on its high levels and early release into the circulation post-MI, C0C1f may serve as a novel biomarker for diagnosing MI more effectively than current clinically used biomarkers. Over time, circulating C0C1f could trigger an autoimmune response leading to myocarditis and progressive cardiac dysfunction. Given the importance of cMyBP-C phosphorylation state in the context of proteolytic cleavage and release into the circulation post-MI, understanding the posttranslational modifications (PTMs) of cMyBP-C would help in further elucidating the role of this protein in health and disease. Accordingly, recent studies have implemented the latest proteomics approaches to define the PTMs of cMyBP-C. The use of such proteomics assays may provide accurate quantitation of the levels of cMyBP-C in the circulation following MI, which could, in turn, demonstrate the efficacy of using plasma cMyBP-C as a cardiac-specific early biomarker of MI. In this review, we define the pathogenic and potential immunogenic effects of C0C1f on cardiac function in the post-MI heart. We also discuss the most advanced proteomics approaches now used to determine cMyBP-C PTMs with the aim of validating C0C1f as an early biomarker of MI. © The Authors PROTEOMICS - Clinical Applications Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Proteomics Is Analytical Chemistry: Fitness-for-Purpose in the Application of Top-Down and Bottom-Up Analyses.

PubMed

Coorssen, Jens R; Yergey, Alfred L

2015-12-03

Molecular mechanisms underlying health and disease function at least in part based on the flexibility and fine-tuning afforded by protein isoforms and post-translational modifications. The ability to effectively and consistently resolve these protein species or proteoforms, as well as assess quantitative changes is therefore central to proteomic analyses. Here we discuss the pros and cons of currently available and developing analytical techniques from the perspective of the full spectrum of available tools and their current applications, emphasizing the concept of fitness-for-purpose in experimental design based on consideration of sample size and complexity; this necessarily also addresses analytical reproducibility and its variance. Data quality is considered the primary criterion, and we thus emphasize that the standards of Analytical Chemistry must apply throughout any proteomic analysis.

Spatial-Resolution Cell Type Proteome Profiling of Cancer Tissue by Fully Integrated Proteomics Technology.

PubMed

Xu, Ruilian; Tang, Jun; Deng, Quantong; He, Wan; Sun, Xiujie; Xia, Ligang; Cheng, Zhiqiang; He, Lisheng; You, Shuyuan; Hu, Jintao; Fu, Yuxiang; Zhu, Jian; Chen, Yixin; Gao, Weina; He, An; Guo, Zhengyu; Lin, Lin; Li, Hua; Hu, Chaofeng; Tian, Ruijun

2018-05-01

Increasing attention has been focused on cell type proteome profiling for understanding the heterogeneous multicellular microenvironment in tissue samples. However, current cell type proteome profiling methods need large amounts of starting materials which preclude their application to clinical tumor specimens with limited access. Here, by seamlessly combining laser capture microdissection and integrated proteomics sample preparation technology SISPROT, specific cell types in tumor samples could be precisely dissected with single cell resolution and processed for high-sensitivity proteome profiling. Sample loss and contamination due to the multiple transfer steps are significantly reduced by the full integration and noncontact design. H&E staining dyes which are necessary for cell type investigation could be selectively removed by the unique two-stage design of the spintip device. This easy-to-use proteome profiling technology achieved high sensitivity with the identification of more than 500 proteins from only 0.1 mm 2 and 10 μm thickness colon cancer tissue section. The first cell type proteome profiling of four cell types from one colon tumor and surrounding normal tissue, including cancer cells, enterocytes, lymphocytes, and smooth muscle cells, was obtained. 5271, 4691, 4876, and 2140 protein groups were identified, respectively, from tissue section of only 5 mm 2 and 10 μm thickness. Furthermore, spatially resolved proteome distribution profiles of enterocytes, lymphocytes, and smooth muscle cells on the same tissue slices and across four consecutive sections with micrometer distance were successfully achieved. This fully integrated proteomics technology, termed LCM-SISPROT, is therefore promising for spatial-resolution cell type proteome profiling of tumor microenvironment with a minute amount of clinical starting materials.

Proteostasis and REDOX state in the heart

PubMed Central

Christians, Elisabeth S.

2012-01-01

Force-generating contractile cells of the myocardium must achieve and maintain their primary function as an efficient mechanical pump over the life span of the organism. Because only half of the cardiomyocytes can be replaced during the entire human life span, the maintenance strategy elicited by cardiac cells relies on uninterrupted renewal of their components, including proteins whose specialized functions constitute this complex and sophisticated contractile apparatus. Thus cardiac proteins are continuously synthesized and degraded to ensure proteome homeostasis, also termed “proteostasis.” Once synthesized, proteins undergo additional folding, posttranslational modifications, and trafficking and/or become involved in protein-protein or protein-DNA interactions to exert their functions. This includes key transient interactions of cardiac proteins with molecular chaperones, which assist with quality control at multiple levels to prevent misfolding or to facilitate degradation. Importantly, cardiac proteome maintenance depends on the cellular environment and, in particular, the reduction-oxidation (REDOX) state, which is significantly different among cardiac organelles (e.g., mitochondria and endoplasmic reticulum). Taking into account the high metabolic activity for oxygen consumption and ATP production by mitochondria, it is a challenge for cardiac cells to maintain the REDOX state while preventing either excessive oxidative or reductive stress. A perturbed REDOX environment can affect protein handling and conformation (e.g., disulfide bonds), disrupt key structure-function relationships, and trigger a pathogenic cascade of protein aggregation, decreased cell survival, and increased organ dysfunction. This review covers current knowledge regarding the general domain of REDOX state and protein folding, specifically in cardiomyocytes under normal-healthy conditions and during disease states associated with morbidity and mortality in humans. PMID:22003057

Proteostasis and REDOX state in the heart.

PubMed

Christians, Elisabeth S; Benjamin, Ivor J

2012-01-01

Force-generating contractile cells of the myocardium must achieve and maintain their primary function as an efficient mechanical pump over the life span of the organism. Because only half of the cardiomyocytes can be replaced during the entire human life span, the maintenance strategy elicited by cardiac cells relies on uninterrupted renewal of their components, including proteins whose specialized functions constitute this complex and sophisticated contractile apparatus. Thus cardiac proteins are continuously synthesized and degraded to ensure proteome homeostasis, also termed "proteostasis." Once synthesized, proteins undergo additional folding, posttranslational modifications, and trafficking and/or become involved in protein-protein or protein-DNA interactions to exert their functions. This includes key transient interactions of cardiac proteins with molecular chaperones, which assist with quality control at multiple levels to prevent misfolding or to facilitate degradation. Importantly, cardiac proteome maintenance depends on the cellular environment and, in particular, the reduction-oxidation (REDOX) state, which is significantly different among cardiac organelles (e.g., mitochondria and endoplasmic reticulum). Taking into account the high metabolic activity for oxygen consumption and ATP production by mitochondria, it is a challenge for cardiac cells to maintain the REDOX state while preventing either excessive oxidative or reductive stress. A perturbed REDOX environment can affect protein handling and conformation (e.g., disulfide bonds), disrupt key structure-function relationships, and trigger a pathogenic cascade of protein aggregation, decreased cell survival, and increased organ dysfunction. This review covers current knowledge regarding the general domain of REDOX state and protein folding, specifically in cardiomyocytes under normal-healthy conditions and during disease states associated with morbidity and mortality in humans.

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.

Exploring the context of the lung proteome within the airway mucosa following allergen challenge.

PubMed

Fehniger, Thomas E; Sato-Folatre, José-Gabriel; Malmström, Johan; Berglund, Magnus; Lindberg, Claes; Brange, Charlotte; Lindberg, Henrik; Marko-Varga, György

2004-01-01

The lung proteome is a dynamic collection of specialized proteins related to pulmonary function. Many cells of different derivations, activation states, and levels of maturity contribute to the changing environment, which produces the lung proteome. Inflammatory cells reacting to environmental challenge, for example from allergens, produce and secrete proteins which have profound effects on both resident and nonresident cells located in airways, alveoli, and the vascular tree which provides blood cells to the parenchyma alveolar bed for gas exchange. In an experimental model of allergic airway inflammation, we have compared control and allergen challenged lung compartments to determine global protein expression patterns using 2D-gel electrophoresis and subsequent spot identification by MS/MS mass spectrometry. We have then specifically isolated the epithelial mucosal layer, which lines conducting airways, from control and allergen challenged lungs, using laser capture technology and performed proteome identification on these selected cell samples. A central component of our investigations has been to contextually relate the histological features of the dynamic pulmonary environment to the changes in protein expression observed following challenge. Our results provide new information of the complexity of the submucosa/epithelium interface and the mechanisms behind the transformation of airway epithelium from normal steady states to functionally activated states.

To label or not to label: applications of quantitative proteomics in neuroscience research.

PubMed

Filiou, Michaela D; Martins-de-Souza, Daniel; Guest, Paul C; Bahn, Sabine; Turck, Christoph W

2012-02-01

Proteomics has provided researchers with a sophisticated toolbox of labeling-based and label-free quantitative methods. These are now being applied in neuroscience research where they have already contributed to the elucidation of fundamental mechanisms and the discovery of candidate biomarkers. In this review, we evaluate and compare labeling-based and label-free quantitative proteomic techniques for applications in neuroscience research. We discuss the considerations required for the analysis of brain and central nervous system specimens, the experimental design of quantitative proteomic workflows as well as the feasibility, advantages, and disadvantages of the available techniques for neuroscience-oriented questions. Furthermore, we assess the use of labeled standards as internal controls for comparative studies in humans and review applications of labeling-based and label-free mass spectrometry approaches in relevant model organisms and human subjects. Providing a comprehensive guide of feasible and meaningful quantitative proteomic methodologies for neuroscience research is crucial not only for overcoming current limitations but also for gaining useful insights into brain function and translating proteomics from bench to bedside. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Translational Research and Plasma Proteomic in Cancer.

PubMed

Santini, Annamaria Chiara; Giovane, Giancarlo; Auletta, Adelaide; Di Carlo, Angelina; Fiorelli, Alfonso; Cito, Letizia; Astarita, Carlo; Giordano, Antonio; Alfano, Roberto; Feola, Antonia; Di Domenico, Marina

2016-04-01

Proteomics is a recent field of research in molecular biology that can help in the fight against cancer through the search for biomarkers that can detect this disease in the early stages of its development. Proteomic is a speedily growing technology, also thanks to the development of even more sensitive and fast mass spectrometry analysis. Although this technique is the most widespread for the discovery of new cancer biomarkers, it still suffers of a poor sensitivity and insufficient reproducibility, essentially due to the tumor heterogeneity. Common technical shortcomings include limitations in the sensitivity of detecting low abundant biomarkers and possible systematic biases in the observed data. Current research attempts are trying to develop high-resolution proteomic instrumentation for high-throughput monitoring of protein changes that occur in cancer. In this review, we describe the basic features of the proteomic tools which have proven to be useful in cancer research, showing their advantages and disadvantages. The application of these proteomic tools could provide early biomarkers detection in various cancer types and could improve the understanding the mechanisms of tumor growth and dissemination. © 2015 Wiley Periodicals, Inc.

Current Progress in Tonoplast Proteomics Reveals Insights into the Function of the Large Central Vacuole

PubMed Central

Trentmann, Oliver; Haferkamp, Ilka

2013-01-01

Vacuoles of plants fulfill various biologically important functions, like turgor generation and maintenance, detoxification, solute sequestration, or protein storage. Different types of plant vacuoles (lytic versus protein storage) are characterized by different functional properties apparently caused by a different composition/abundance and regulation of transport proteins in the surrounding membrane, the tonoplast. Proteome analyses allow the identification of vacuolar proteins and provide an informative basis for assigning observed transport processes to specific carriers or channels. This review summarizes techniques required for vacuolar proteome analyses, like e.g., isolation of the large central vacuole or tonoplast membrane purification. Moreover, an overview about diverse published vacuolar proteome studies is provided. It becomes evident that qualitative proteomes from different plant species represent just the tip of the iceberg. During the past few years, mass spectrometry achieved immense improvement concerning its accuracy, sensitivity, and application. As a consequence, modern tonoplast proteome approaches are suited for detecting alterations in membrane protein abundance in response to changing environmental/physiological conditions and help to clarify the regulation of tonoplast transport processes. PMID:23459586

Elucidation of salt stress defense and tolerance mechanisms of crop plants using proteomics--current achievements and perspectives.

PubMed

Barkla, Bronwyn J; Castellanos-Cervantes, Thelma; de León, José L Diaz; Matros, Andrea; Mock, Hans-Peter; Perez-Alfocea, Francisco; Salekdeh, Ghasem H; Witzel, Katja; Zörb, Christian

2013-06-01

Salinity is a major threat limiting the productivity of crop plants. A clear demand for improving the salinity tolerance of the major crop plants is imposed by the rapidly growing world population. This review summarizes the achievements of proteomic studies to elucidate the response mechanisms of selected model and crop plants to cope with salinity stress. We also aim at identifying research areas, which deserve increased attention in future proteome studies, as a prerequisite to identify novel targets for breeding strategies. Such areas include the impact of plant-microbial communities on the salinity tolerance of crops under field conditions, the importance of hormone signaling in abiotic stress tolerance, and the significance of control mechanisms underlying the observed changes in the proteome patterns. We briefly highlight the impact of novel tools for future proteome studies and argue for the use of integrated approaches. The evaluation of genetic resources by means of novel automated phenotyping facilities will have a large impact on the application of proteomics especially in combination with metabolomics or transcriptomics. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomics in investigation of cancer metastasis: functional and clinical consequences and methodological challenges.

PubMed

Maryáš, Josef; Faktor, Jakub; Dvořáková, Monika; Struhárová, Iva; Grell, Peter; Bouchal, Pavel

2014-03-01

Metastases are responsible for most of the cases of death in patients with solid tumors. There is thus an urgent clinical need of better understanding the exact molecular mechanisms and finding novel therapeutics targets and biomarkers of metastatic disease of various tumors. Metastases are formed in a complicated biological process called metastatic cascade. Up to now, proteomics has enabled the identification of number of metastasis-associated proteins and potential biomarkers in cancer tissues, microdissected cells, model systems, and secretomes. Expression profiles and biological role of key proteins were confirmed in verification and functional experiments. This communication reviews these observations and analyses the methodological aspects of the proteomics approaches used. Moreover, it reviews contribution of current proteomics in the field of functional characterization and interactome analysis of proteins involved in various events in metastatic cascade. It is evident that ongoing technical progress will further increase proteome coverage and sample capacity of proteomics technologies, giving complex answers to clinical and functional questions asked. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

MCAM: multiple clustering analysis methodology for deriving hypotheses and insights from high-throughput proteomic datasets.

PubMed

Naegle, Kristen M; Welsch, Roy E; Yaffe, Michael B; White, Forest M; Lauffenburger, Douglas A

2011-07-01

Advances in proteomic technologies continue to substantially accelerate capability for generating experimental data on protein levels, states, and activities in biological samples. For example, studies on receptor tyrosine kinase signaling networks can now capture the phosphorylation state of hundreds to thousands of proteins across multiple conditions. However, little is known about the function of many of these protein modifications, or the enzymes responsible for modifying them. To address this challenge, we have developed an approach that enhances the power of clustering techniques to infer functional and regulatory meaning of protein states in cell signaling networks. We have created a new computational framework for applying clustering to biological data in order to overcome the typical dependence on specific a priori assumptions and expert knowledge concerning the technical aspects of clustering. Multiple clustering analysis methodology ('MCAM') employs an array of diverse data transformations, distance metrics, set sizes, and clustering algorithms, in a combinatorial fashion, to create a suite of clustering sets. These sets are then evaluated based on their ability to produce biological insights through statistical enrichment of metadata relating to knowledge concerning protein functions, kinase substrates, and sequence motifs. We applied MCAM to a set of dynamic phosphorylation measurements of the ERRB network to explore the relationships between algorithmic parameters and the biological meaning that could be inferred and report on interesting biological predictions. Further, we applied MCAM to multiple phosphoproteomic datasets for the ERBB network, which allowed us to compare independent and incomplete overlapping measurements of phosphorylation sites in the network. We report specific and global differences of the ERBB network stimulated with different ligands and with changes in HER2 expression. Overall, we offer MCAM as a broadly-applicable approach for analysis of proteomic data which may help increase the current understanding of molecular networks in a variety of biological problems. © 2011 Naegle et al.

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.

Synovial fluid proteomics in the pursuit of arthritis mediators: An evolving field of novel biomarker discovery.

PubMed

Mahendran, Shalini M; Oikonomopoulou, Katerina; Diamandis, Eleftherios P; Chandran, Vinod

Synovial fluid (SF) is a protein-rich fluid produced into the joint cavity by cells of the synovial membrane. Due to its direct contact with articular cartilage, surfaces of the bone, and the synoviocytes of the inner membrane, it provides a promising reflection of the biochemical state of the joint under varying physiological and pathophysiological conditions. This property of SF has been exploited within numerous studies in search of unique biomarkers of joint pathologies with the ultimate goal of developing minimally invasive clinical assays to detect and/or monitor disease states. Several proteomic methodologies have been employed to mine the SF proteome. From elementary immunoassays to high-throughput analyses using mass spectrometry-based techniques, each has demonstrated distinct advantages and disadvantages in the identification and quantification of SF proteins. This review will explore the role of SF in the elucidation of the arthritis proteome and the extent to which high-throughput techniques have facilitated the discovery and validation of protein biomarkers from osteoarthritis (OA), rheumatoid arthritis (RA), psoriatic arthritis (PsA), and juvenile idiopathic arthritis (JIA) patients.

Methods for evaluating the Caenorhabditis elegans dauer state: standard dauer-formation assay using synthetic daumones and proteomic analysis of O-GlcNAc modifications.

PubMed

Lee, Jeeyong; Kim, Kwang-Youl; Joo, Hyoe-Jin; Kim, Heekyeong; Jeong, Pan-Young; Paik, Young-Ki

2011-01-01

The dauer state is a non-feeding, alternative L3 state characterized by a number of distinctive metabolic and morphological changes. There are many naturally occurring dauer-inducing pheromones, termed daumones, that have been suggested by some to exhibit differences in dauer-inducing activity. Here, we have established a standard dauer-formation assay that uses synthetic daumones 1, 2, and 3, the three major daumones. To analyze the proteome of Caenorhabditis elegans in the dauer state, we focused on O-GlcNAc modification, a cytosolic modification of proteins that is known to interact either competitively or synergistically with protein phosphorylation. Protein O-GlcNAc modification is an important biological process in cells that can ensure the timely response to extracellular stimuli, such as daumone, and maintain cellular homeostasis. Establishing a standard method for assaying dauer formation using different synthetic daumones, and using differences in O-GlcNAcylated proteins during the dauer state to analyze the dauer proteome will lead to a better understanding of dauer biology of C. elegans in the context of animal longevity and adaptation under harsh environments. Copyright © 2011 Elsevier Inc. 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.

The ProteomeXchange consortium in 2017: supporting the cultural change in proteomics public data deposition

PubMed Central

Deutsch, Eric W.; Csordas, Attila; Sun, Zhi; Jarnuczak, Andrew; Perez-Riverol, Yasset; Ternent, Tobias; Campbell, David S.; Bernal-Llinares, Manuel; Okuda, Shujiro; Kawano, Shin; Moritz, Robert L.; Carver, Jeremy J.; Wang, Mingxun; Ishihama, Yasushi; Bandeira, Nuno; Hermjakob, Henning; Vizcaíno, Juan Antonio

2017-01-01

The ProteomeXchange (PX) Consortium of proteomics resources (http://www.proteomexchange.org) was formally started in 2011 to standardize data submission and dissemination of mass spectrometry proteomics data worldwide. We give an overview of the current consortium activities and describe the advances of the past few years. Augmenting the PX founding members (PRIDE and PeptideAtlas, including the PASSEL resource), two new members have joined the consortium: MassIVE and jPOST. ProteomeCentral remains as the common data access portal, providing the ability to search for data sets in all participating PX resources, now with enhanced data visualization components. We describe the updated submission guidelines, now expanded to include four members instead of two. As demonstrated by data submission statistics, PX is supporting a change in culture of the proteomics field: public data sharing is now an accepted standard, supported by requirements for journal submissions resulting in public data release becoming the norm. More than 4500 data sets have been submitted to the various PX resources since 2012. Human is the most represented species with approximately half of the data sets, followed by some of the main model organisms and a growing list of more than 900 diverse species. Data reprocessing activities are becoming more prominent, with both MassIVE and PeptideAtlas releasing the results of reprocessed data sets. Finally, we outline the upcoming advances for ProteomeXchange. PMID:27924013

Updated biological roles for matrix metalloproteinases and new "intracellular" substrates revealed by degradomics.

PubMed

Butler, Georgina S; Overall, Christopher M

2009-11-24

Shotgun proteomics techniques are conceptually unbiased, but data interpretation and follow-up experiments are often constrained by dogma, established beliefs that are accepted without question, that can dilute the power of proteomics and hinder scientific progress. Proteomics and degradomics, the characterization of all proteases, inhibitors, and protease substrates by genomic and proteomic techniques, have exponentially expanded the known substrate repertoire of the matrix metalloproteinases (MMPs), even to include intracellular proteins with newly recognized extracellular functions. Thus, the dogma that MMPs are dowdy degraders of extracellular matrix has been resolutely overturned, and the metamorphosis of MMPs into modulators of multiple signaling pathways has been facilitated. Here we review progress made in the field of degradomics and present a current view of the MMP degradome.

Combining genomic and proteomic approaches for epigenetics research

PubMed Central

Han, Yumiao; Garcia, Benjamin A

2014-01-01

Epigenetics is the study of changes in gene expression or cellular phenotype that do not change the DNA sequence. In this review, current methods, both genomic and proteomic, associated with epigenetics research are discussed. Among them, chromatin immunoprecipitation (ChIP) followed by sequencing and other ChIP-based techniques are powerful techniques for genome-wide profiling of DNA-binding proteins, histone post-translational modifications or nucleosome positions. However, mass spectrometry-based proteomics is increasingly being used in functional biological studies and has proved to be an indispensable tool to characterize histone modifications, as well as DNA–protein and protein–protein interactions. With the development of genomic and proteomic approaches, combination of ChIP and mass spectrometry has the potential to expand our knowledge of epigenetics research to a higher level. PMID:23895656

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.

The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides

PubMed Central

Tsirigos, Konstantinos D.; Peters, Christoph; Shu, Nanjiang; Käll, Lukas; Elofsson, Arne

2015-01-01

TOPCONS (http://topcons.net/) is a widely used web server for consensus prediction of membrane protein topology. We hereby present a major update to the server, with some substantial improvements, including the following: (i) TOPCONS can now efficiently separate signal peptides from transmembrane regions. (ii) The server can now differentiate more successfully between globular and membrane proteins. (iii) The server now is even slightly faster, although a much larger database is used to generate the multiple sequence alignments. For most proteins, the final prediction is produced in a matter of seconds. (iv) The user-friendly interface is retained, with the additional feature of submitting batch files and accessing the server programmatically using standard interfaces, making it thus ideal for proteome-wide analyses. Indicatively, the user can now scan the entire human proteome in a few days. (v) For proteins with homology to a known 3D structure, the homology-inferred topology is also displayed. (vi) Finally, the combination of methods currently implemented achieves an overall increase in performance by 4% as compared to the currently available best-scoring methods and TOPCONS is the only method that can identify signal peptides and still maintain a state-of-the-art performance in topology predictions. PMID:25969446

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.

Exploring novel paths towards protein signatures of chronic pain.

PubMed

Gomez-Varela, David; Schmidt, Manuela

2016-01-01

Pain is a major symptom of many medical conditions and the worldwide number one reason for people to seek medical assistance. It affects the quality of life of patients and poses a heavy financial burden on society with high costs of treatment and lost productivity. Furthermore, the treatment of chronic pain presents a big challenge as pain therapeutics often lack efficacy and exhibit minimal safety profiles. The latter can be largely attributed to the fact that current therapies target molecules with key physiological functions throughout the body. In light of these difficulties, the identification of proteins specifically involved in chronic pain states is of paramount importance for designing selective interventions. Several profiling efforts have been employed with the aim to dissect the molecular underpinnings of chronic pain, both on the level of the transcriptome and proteome. However, generated results are often inconsistent and non-overlapping, which is largely due to inherent technical constraints. A potential solution may be offered by emerging strategies capable of performing standardized and reproducible proteome analysis, such as data-independent acquisition-mass spectrometry (DIA-MS). We have recently demonstrated the applicability of DIA-MS to interrogate chronic pain-related proteome alterations in mice. Based on our results, we aim to provide an overview on DIA-MS and its potential to contribute to the comprehensive characterization of molecular signatures underlying pain pathologies. © The Author(s) 2016.

An inventory of the Aspergillus niger secretome by combining in silico predictions with shotgun proteomics data.

PubMed

Braaksma, Machtelt; Martens-Uzunova, Elena S; Punt, Peter J; Schaap, Peter J

2010-10-19

The ecological niche occupied by a fungal species, its pathogenicity and its usefulness as a microbial cell factory to a large degree depends on its secretome. Protein secretion usually requires the presence of a N-terminal signal peptide (SP) and by scanning for this feature using available highly accurate SP-prediction tools, the fraction of potentially secreted proteins can be directly predicted. However, prediction of a SP does not guarantee that the protein is actually secreted and current in silico prediction methods suffer from gene-model errors introduced during genome annotation. A majority rule based classifier that also evaluates signal peptide predictions from the best homologs of three neighbouring Aspergillus species was developed to create an improved list of potential signal peptide containing proteins encoded by the Aspergillus niger genome. As a complement to these in silico predictions, the secretome associated with growth and upon carbon source depletion was determined using a shotgun proteomics approach. Overall, some 200 proteins with a predicted signal peptide were identified to be secreted proteins. Concordant changes in the secretome state were observed as a response to changes in growth/culture conditions. Additionally, two proteins secreted via a non-classical route operating in A. niger were identified. We were able to improve the in silico inventory of A. niger secretory proteins by combining different gene-model predictions from neighbouring Aspergilli and thereby avoiding prediction conflicts associated with inaccurate gene-models. The expected accuracy of signal peptide prediction for proteins that lack homologous sequences in the proteomes of related species is 85%. An experimental validation of the predicted proteome confirmed in silico predictions.

An inventory of the Aspergillus niger secretome by combining in silico predictions with shotgun proteomics data

PubMed Central

2010-01-01

Background The ecological niche occupied by a fungal species, its pathogenicity and its usefulness as a microbial cell factory to a large degree depends on its secretome. Protein secretion usually requires the presence of a N-terminal signal peptide (SP) and by scanning for this feature using available highly accurate SP-prediction tools, the fraction of potentially secreted proteins can be directly predicted. However, prediction of a SP does not guarantee that the protein is actually secreted and current in silico prediction methods suffer from gene-model errors introduced during genome annotation. Results A majority rule based classifier that also evaluates signal peptide predictions from the best homologs of three neighbouring Aspergillus species was developed to create an improved list of potential signal peptide containing proteins encoded by the Aspergillus niger genome. As a complement to these in silico predictions, the secretome associated with growth and upon carbon source depletion was determined using a shotgun proteomics approach. Overall, some 200 proteins with a predicted signal peptide were identified to be secreted proteins. Concordant changes in the secretome state were observed as a response to changes in growth/culture conditions. Additionally, two proteins secreted via a non-classical route operating in A. niger were identified. Conclusions We were able to improve the in silico inventory of A. niger secretory proteins by combining different gene-model predictions from neighbouring Aspergilli and thereby avoiding prediction conflicts associated with inaccurate gene-models. The expected accuracy of signal peptide prediction for proteins that lack homologous sequences in the proteomes of related species is 85%. An experimental validation of the predicted proteome confirmed in silico predictions. PMID:20959013

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.

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.

Secretome of Aspergillus oryzae in Shaoxing rice wine koji.

PubMed

Zhang, Bo; Guan, Zheng-Bing; Cao, Yu; Xie, Guang-Fa; Lu, Jian

2012-04-16

Shaoxing rice wine is the most famous and representative Chinese rice wine. Aspergillus oryzae SU16 is used in the manufacture of koji, the Shaoxing rice wine starter culture. In the current study, a comprehensive analysis of the secretome profile of A. oryzae SU16 in Shaoxing rice wine koji was performed for the first time. The proteomic analysis for the identification of the secretory proteins was done using two-dimensional electrophoresis combined with matrix-assisted laser desorption/ionization-tandem time of flight mass spectrometry based on the annotated A. oryzae genome sequence. A total of 41 unique proteins were identified from the secretome. These proteins included 17 extracellular proteins following the classical secretory pathway, and 10 extracellular proteins putatively secreted by the non-classical secretory pathway. The present secretome profile greatly differed from previous reports on A. oryzae growing in other solid-state nutrient sources. Several new secretory or putative secretory proteins were also found. These proteomic data will significantly aid the advancement of research on the secretome of A. oryzae, especially in solid-state cultures, and in elucidating the production process mechanism of Shaoxing rice wine koji. The findings may promote the technological development and innovation of the Shaoxing rice wine industry. Copyright © 2012 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

Proteomic study of acute respiratory distress syndrome: current knowledge and implications for drug development

PubMed Central

Levitt, Joseph E.; Rogers, Angela J.

2017-01-01

The acute respiratory distress syndrome (ARDS) is a common cause of acute respiratory failure, and is associated with substantial mortality and morbidity. Dozens of clinical trials targeting ARDS have failed, with no drug specifically targeting lung injury in widespread clinical use. Thus, the need for drug development in ARDS is great. Targeted proteomic studies in ARDS have identified many key pathways in the disease, including inflammation, epithelial injury, endothelial injury or activation, and disordered coagulation and repair. Recent studies reveal the potential for proteomic changes to identify novel subphenotypes of ARDS patients who may be most likely to respond to therapy and could thus be targeted for enrollment in clinical trials. Nontargeted studies of proteomics in ARDS are just beginning and have the potential to identify novel drug targets and key pathways in the disease. Proteomics will play an important role in phenotyping of patients and developing novel therapies for ARDS in the future. PMID:27031735

Proteomics meets blue biotechnology: a wealth of novelties and opportunities.

PubMed

Hartmann, Erica M; Durighello, Emie; Pible, Olivier; Nogales, Balbina; Beltrametti, Fabrizio; Bosch, Rafael; Christie-Oleza, Joseph A; Armengaud, Jean

2014-10-01

Blue biotechnology, in which aquatic environments provide the inspiration for various products such as food additives, aquaculture, biosensors, green chemistry, bioenergy, and pharmaceuticals, holds enormous promise. Large-scale efforts to sequence aquatic genomes and metagenomes, as well as campaigns to isolate new organisms and culture-based screenings, are helping to push the boundaries of known organisms. Mass spectrometry-based proteomics can complement 16S gene sequencing in the effort to discover new organisms of potential relevance to blue biotechnology by facilitating the rapid screening of microbial isolates and by providing in depth profiles of the proteomes and metaproteomes of marine organisms, both model cultivable isolates and, more recently, exotic non-cultivable species and communities. Proteomics has already contributed to blue biotechnology by identifying aquatic proteins with potential applications to food fermentation, the textile industry, and biomedical drug development. In this review, we discuss historical developments in blue biotechnology, the current limitations to the known marine biosphere, and the ways in which mass spectrometry can expand that knowledge. We further speculate about directions that research in blue biotechnology will take given current and near-future technological advancements in mass spectrometry. Copyright © 2014 Elsevier B.V. All rights reserved.

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

Proteomics of Skeletal Muscle: Focus on Insulin Resistance and Exercise Biology

PubMed Central

Deshmukh, Atul S.

2016-01-01

Skeletal muscle is the largest tissue in the human body and plays an important role in locomotion and whole body metabolism. It accounts for ~80% of insulin stimulated glucose disposal. Skeletal muscle insulin resistance, a primary feature of Type 2 diabetes, is caused by a decreased ability of muscle to respond to circulating insulin. Physical exercise improves insulin sensitivity and whole body metabolism and remains one of the most promising interventions for the prevention of Type 2 diabetes. Insulin resistance and exercise adaptations in skeletal muscle might be a cause, or consequence, of altered protein expressions profiles and/or their posttranslational modifications (PTMs). Mass spectrometry (MS)-based proteomics offer enormous promise for investigating the molecular mechanisms underlying skeletal muscle insulin resistance and exercise-induced adaptation; however, skeletal muscle proteomics are challenging. This review describes the technical limitations of skeletal muscle proteomics as well as emerging developments in proteomics workflow with respect to samples preparation, liquid chromatography (LC), MS and computational analysis. These technologies have not yet been fully exploited in the field of skeletal muscle proteomics. Future studies that involve state-of-the-art proteomics technology will broaden our understanding of exercise-induced adaptations as well as molecular pathogenesis of insulin resistance. This could lead to the identification of new therapeutic targets. PMID:28248217

Neural Stem Cells (NSCs) and Proteomics*

PubMed Central

Shoemaker, Lorelei D.; Kornblum, Harley I.

2016-01-01

Neural stem cells (NSCs) can self-renew and give rise to the major cell types of the CNS. Studies of NSCs include the investigation of primary, CNS-derived cells as well as animal and human embryonic stem cell (ESC)-derived and induced pluripotent stem cell (iPSC)-derived sources. NSCs provide a means with which to study normal neural development, neurodegeneration, and neurological disease and are clinically relevant sources for cellular repair to the damaged and diseased CNS. Proteomics studies of NSCs have the potential to delineate molecules and pathways critical for NSC biology and the means by which NSCs can participate in neural repair. In this review, we provide a background to NSC biology, including the means to obtain them and the caveats to these processes. We then focus on advances in the proteomic interrogation of NSCs. This includes the analysis of posttranslational modifications (PTMs); approaches to analyzing different proteomic compartments, such the secretome; as well as approaches to analyzing temporal differences in the proteome to elucidate mechanisms of differentiation. We also discuss some of the methods that will undoubtedly be useful in the investigation of NSCs but which have not yet been applied to the field. While many proteomics studies of NSCs have largely catalogued the proteome or posttranslational modifications of specific cellular states, without delving into specific functions, some have led to understandings of functional processes or identified markers that could not have been identified via other means. Many challenges remain in the field, including the precise identification and standardization of NSCs used for proteomic analyses, as well as how to translate fundamental proteomics studies to functional biology. The next level of investigation will require interdisciplinary approaches, combining the skills of those interested in the biochemistry of proteomics with those interested in modulating NSC function. PMID:26494823

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.

Platelet proteomics: from discovery to diagnosis.

PubMed

Looße, Christina; Swieringa, Frauke; Heemskerk, Johan W M; Sickmann, Albert; Lorenz, Christin

2018-05-22

Platelets are the smallest cells within the circulating blood with key roles in physiological haemostasis and pathological thrombosis regulated by the onset of activating/inhibiting processes via receptor responses and signalling cascades. Areas covered: Proteomics as well as genomic approaches have been fundamental in identifying and quantifying potential targets for future diagnostic strategies in the prevention of bleeding and thrombosis, and uncovering the complexity of platelet functions in health and disease. In this article, we provide a critical overview on current functional tests used in diagnostics and the future perspectives for platelet proteomics in clinical applications. Expert commentary: Proteomics represents a valuable tool for the identification of patients with diverse platelet associated defects. In-depth validation of identified biomarkers, e.g. receptors, signalling proteins, post-translational modifications, in large cohorts is decisive for translation into routine clinical diagnostics.

Proteomics in pharmaceutical research and development.

PubMed

Cutler, Paul; Voshol, Hans

2015-08-01

In the 20 years since its inception, the evolution of proteomics in pharmaceutical industry has mirrored the developments within academia and indeed other industries. From initial enthusiasm and subsequent disappointment in global protein expression profiling, pharma research saw the biggest impact when relating to more focused approaches, such as those exploring the interaction between proteins and drugs. Nowadays, proteomics technologies have been integrated in many areas of pharmaceutical R&D, ranging from the analysis of therapeutic proteins to the monitoring of clinical trials. Here, we review the development of proteomics in the drug discovery process, placing it in a historical context as well as reviewing the current status in light of the contributions to this special issue, which reflect some of the diverse demands of the drug and biomarker pipelines. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A multi-protease, multi-dissociation, bottom-up-to-top-down proteomic view of the Loxosceles intermedia venom

PubMed Central

Trevisan-Silva, Dilza; Bednaski, Aline V.; Fischer, Juliana S.G.; Veiga, Silvio S.; Bandeira, Nuno; Guthals, Adrian; Marchini, Fabricio K.; Leprevost, Felipe V.; Barbosa, Valmir C.; Senff-Ribeiro, Andrea; Carvalho, Paulo C.

2017-01-01

Venoms are a rich source for the discovery of molecules with biotechnological applications, but their analysis is challenging even for state-of-the-art proteomics. Here we report on a large-scale proteomic assessment of the venom of Loxosceles intermedia, the so-called brown spider. Venom was extracted from 200 spiders and fractioned into two aliquots relative to a 10 kDa cutoff mass. Each of these was further fractioned and digested with trypsin (4 h), trypsin (18 h), pepsin (18 h), and chymotrypsin (18 h), then analyzed by MudPIT on an LTQ-Orbitrap XL ETD mass spectrometer fragmenting precursors by CID, HCD, and ETD. Aliquots of undigested samples were also analyzed. Our experimental design allowed us to apply spectral networks, thus enabling us to obtain meta-contig assemblies, and consequently de novo sequencing of practically complete proteins, culminating in a deep proteome assessment of the venom. Data are available via ProteomeXchange, with identifier PXD005523. PMID:28696408

Clinical proteomics: Applications for prostate cancer biomarker discovery and detection.

PubMed

Petricoin, Emanuel F; Ornstein, David K; Liotta, Lance A

2004-01-01

The science of proteomics comprises much more than simply generating lists of proteins that change in expression as a cause of or consequence of pathophysiology. The goal of proteomics should be to characterize the information flow through the intercellular protein circuitry that communicates with the extracellular microenvironment and then ultimately to the serum/plasma macroenvironment. Serum proteomic pattern diagnostics is a new type of proteomic concept in which patterns of ion signatures generated from high dimensional mass spectrometry data are used as diagnostic classifiers. This recent approach has exciting potential for clinical utility of diagnostic patterns because low molecular weight metabolites, peptides, and protein fragments may have higher accuracy than traditional biomarkers of cancer detection. Intriguingly, we now have discovered that this diagnostic information exists in a bound state, complexed with circulating highly abundant carrier proteins. These diagnostic fragments may one day be harvested by circulating nanoparticles, designed to absorb, enrich, and amplify the repertoire of diagnostic biomarkers generated-even at the critical, initial stages of carcinogenesis. Copyright 2004 Elsevier Inc.

Recommendations for Mass Spectrometry Data Quality Metrics for Open Access Data (Corollary to the Amsterdam Principles)*

PubMed Central

Kinsinger, Christopher R.; Apffel, James; Baker, Mark; Bian, Xiaopeng; Borchers, Christoph H.; Bradshaw, Ralph; Brusniak, Mi-Youn; Chan, Daniel W.; Deutsch, Eric W.; Domon, Bruno; Gorman, Jeff; Grimm, Rudolf; Hancock, William; Hermjakob, Henning; Horn, David; Hunter, Christie; Kolar, Patrik; Kraus, Hans-Joachim; Langen, Hanno; Linding, Rune; Moritz, Robert L.; Omenn, Gilbert S.; Orlando, Ron; Pandey, Akhilesh; Ping, Peipei; Rahbar, Amir; Rivers, Robert; Seymour, Sean L.; Simpson, Richard J.; Slotta, Douglas; Smith, Richard D.; Stein, Stephen E.; Tabb, David L.; Tagle, Danilo; Yates, John R.; Rodriguez, Henry

2011-01-01

Policies supporting the rapid and open sharing of proteomic data are being implemented by the leading journals in the field. The proteomics community is taking steps to ensure that data are made publicly accessible and are of high quality, a challenging task that requires the development and deployment of methods for measuring and documenting data quality metrics. On September 18, 2010, the United States National Cancer Institute convened the “International Workshop on Proteomic Data Quality Metrics” in Sydney, Australia, to identify and address issues facing the development and use of such methods for open access proteomics data. The stakeholders at the workshop enumerated the key principles underlying a framework for data quality assessment in mass spectrometry data that will meet the needs of the research community, journals, funding agencies, and data repositories. Attendees discussed and agreed up on two primary needs for the wide use of quality metrics: 1) an evolving list of comprehensive quality metrics and 2) standards accompanied by software analytics. Attendees stressed the importance of increased education and training programs to promote reliable protocols in proteomics. This workshop report explores the historic precedents, key discussions, and necessary next steps to enhance the quality of open access data. By agreement, this article is published simultaneously in the Journal of Proteome Research, Molecular and Cellular Proteomics, Proteomics, and Proteomics Clinical Applications as a public service to the research community. The peer review process was a coordinated effort conducted by a panel of referees selected by the journals. PMID:22052993

Uncovering the hidden: complexity and strategies for diagnosing latent tuberculosis.

PubMed

Flores-Valdez, Mario Alberto

2017-10-24

Tuberculosis produces two clinical manifestations: active and latent (non-apparent) disease. The latter is estimated to affect one-third of the world population and constitutes a source of continued transmission should the disease emerge from its hidden state (reactivation). Methods to diagnose latent TB have been evolving and aim to detect the disease in people who are truly infected with M. tuberculosis , versus those where other mycobacteria, or even other pathologies not related to TB, are present. The current use of proteomic and transcriptomic approaches may lead to improved detection methods in the coming years.

Serum proteome profiling in canine idiopathic dilated cardiomyopathy using TMT-based quantitative proteomics approach.

PubMed

Bilić, Petra; Guillemin, Nicolas; Kovačević, Alan; Beer Ljubić, Blanka; Jović, Ines; Galan, Asier; Eckersall, Peter David; Burchmore, Richard; Mrljak, Vladimir

2018-05-15

Idiopathic dilated cardiomyopathy (iDCM) is a primary myocardial disorder with an unknown aetiology, characterized by reduced contractility and ventricular dilation of the left or both ventricles. Naturally occurring canine iDCM was used herein to identify serum proteomic signature of the disease compared to the healthy state, providing an insight into underlying mechanisms and revealing proteins with biomarker potential. To achieve this, we used high-throughput label-based quantitative LC-MS/MS proteomics approach and bioinformatics analysis of the in silico inferred interactome protein network created from the initial list of differential proteins. To complement the proteomic analysis, serum biochemical parameters and levels of know biomarkers of cardiac function were measured. Several proteins with biomarker potential were identified, such as inter-alpha-trypsin inhibitor heavy chain H4, microfibril-associated glycoprotein 4 and apolipoprotein A-IV, which were validated using an independent method (Western blotting) and showed high specificity and sensitivity according to the receiver operating characteristic curve analysis. Bioinformatics analysis revealed involvement of different pathways in iDCM, such as complement cascade activation, lipoprotein particles dynamics, elastic fibre formation, GPCR signalling and respiratory electron transport chain. Idiopathic dilated cardiomyopathy is a severe primary myocardial disease of unknown cause, affecting both humans and dogs. This study is a contribution to the canine heart disease research by means of proteomic and bioinformatic state of the art analyses, following similar approach in human iDCM research. Importantly, we used serum as non-invasive and easily accessible biological source of information and contributed to the scarce data on biofluid proteome research on this topic. Bioinformatics analysis revealed biological pathways modulated in canine iDCM with potential of further targeted research. Also, several proteins with biomarker potential have been identified and successfully validated. Copyright © 2018 Elsevier B.V. All rights reserved.

Advances in microscale separations towards nanoproteomics applications

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

Yi, Lian; Piehowski, Paul D.; Shi, Tujin

Microscale separations (e.g., liquid chromatography or capillary electrophoresis) coupled with mass spectrometry (MS) has become the primary tool for advanced proteomics, an indispensable technology for gaining understanding of complex biological processes. While significant advances have been achieved in MS-based proteomics, the current platforms still face a significant challenge in overall sensitivity towards nanoproteomics (i.e., with less than 1 g total amount of proteins available) applications such as cellular heterogeneity in tissue pathologies. Herein, we review recent advances in microscale separation techniques and integrated sample processing systems that improve the overall sensitivity and coverage of the proteomics workflow, and their contributionsmore » towards nanoproteomics applications.« less

47th Vice President of the United States Joseph R. Biden, Jr. to deliver keynote address at HUPO2017 Global Leadership Gala Dinner | Office of Cancer Clinical Proteomics Research

Cancer.gov

The Human Proteome Organization (HUPO) and Prof Steve Pennington, UCD, chair of the organizing committee of HUPO2017 (the 16th HUPO World Congress) in collaboration with the National Cancer Institute’s (NCI) International Cancer Proteogenome Consortium (ICPC) ann

2D DIGE Does Not Reveal all: A Scotopic Report Suggests Differential Expression of a Single "Calponin Family Member" Protein for Tetany of Sphincters!

PubMed

Chaudhury, Arun

2015-01-01

Using 2D differential gel electrophoresis (DIGE) and mass spectrometry (MS), a recent report by Rattan and Ali (2015) compared proteome expression between tonically contracted sphincteric smooth muscles of the internal anal sphincter (IAS), in comparison to the adjacent rectum [rectal smooth muscles (RSM)] that contracts in a phasic fashion. The study showed the differential expression of a single 23 kDa protein SM22, which was 1.87 fold, overexpressed in RSM in comparison to IAS. Earlier studies have shown differences in expression of different proteins like Rho-associated protein kinase II, myosin light chain kinase, myosin phosphatase, and protein kinase C between IAS and RSM. The currently employed methods, despite its high-throughput potential, failed to identify these well-characterized differences between phasic and tonic muscles. This calls into question the fidelity and validatory potential of the otherwise powerful technology of 2D DIGE/MS. These discrepancies, when redressed in future studies, will evolve this recent report as an important baseline study of "sphincter proteome." Proteomics techniques are currently underutilized in examining pathophysiology of hypertensive/hypotensive disorders involving gastrointestinal sphincters, including achalasia, gastroesophageal reflux disease (GERD), spastic pylorus, seen during diabetes or chronic chemotherapy, intestinal pseudo-obstruction, and recto-anal incontinence. Global proteome mapping may provide instant snapshot of the complete repertoire of differential proteins, thus expediting to identify the molecular pathology of gastrointestinal motility disorders currently labeled "idiopathic" and facilitating practice of precision medicine.

Feasibility of investigating differential proteomic expression in depression: implications for biomarker development in mood disorders

PubMed Central

Frye, M A; Nassan, M; Jenkins, G D; Kung, S; Veldic, M; Palmer, B A; Feeder, S E; Tye, S J; Choi, D S; Biernacka, J M

2015-01-01

The objective of this study was to determine whether proteomic profiling in serum samples can be utilized in identifying and differentiating mood disorders. A consecutive sample of patients with a confirmed diagnosis of unipolar (UP n=52) or bipolar depression (BP-I n=46, BP-II n=49) and controls (n=141) were recruited. A 7.5-ml blood sample was drawn for proteomic multiplex profiling of 320 proteins utilizing the Myriad RBM Discovery Multi-Analyte Profiling platform. After correcting for multiple testing and adjusting for covariates, growth differentiation factor 15 (GDF-15), hemopexin (HPX), hepsin (HPN), matrix metalloproteinase-7 (MMP-7), retinol-binding protein 4 (RBP-4) and transthyretin (TTR) all showed statistically significant differences among groups. In a series of three post hoc analyses correcting for multiple testing, MMP-7 was significantly different in mood disorder (BP-I+BP-II+UP) vs controls, MMP-7, GDF-15, HPN were significantly different in bipolar cases (BP-I+BP-II) vs controls, and GDF-15, HPX, HPN, RBP-4 and TTR proteins were all significantly different in BP-I vs controls. Good diagnostic accuracy (ROC-AUC⩾0.8) was obtained most notably for GDF-15, RBP-4 and TTR when comparing BP-I vs controls. While based on a small sample not adjusted for medication state, this discovery sample with a conservative method of correction suggests feasibility in using proteomic panels to assist in identifying and distinguishing mood disorders, in particular bipolar I disorder. Replication studies for confirmation, consideration of state vs trait serial assays to delineate proteomic expression of bipolar depression vs previous mania, and utility studies to assess proteomic expression profiling as an advanced decision making tool or companion diagnostic are encouraged. PMID:26645624

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.

Consolidation of proteomics data in the Cancer Proteomics database.

PubMed

Arntzen, Magnus Ø; Boddie, Paul; Frick, Rahel; Koehler, Christian J; Thiede, Bernd

2015-11-01

Cancer is a class of diseases characterized by abnormal cell growth and one of the major reasons for human deaths. Proteins are involved in the molecular mechanisms leading to cancer, furthermore they are affected by anti-cancer drugs, and protein biomarkers can be used to diagnose certain cancer types. Therefore, it is important to explore the proteomics background of cancer. In this report, we developed the Cancer Proteomics database to re-interrogate published proteome studies investigating cancer. The database is divided in three sections related to cancer processes, cancer types, and anti-cancer drugs. Currently, the Cancer Proteomics database contains 9778 entries of 4118 proteins extracted from 143 scientific articles covering all three sections: cell death (cancer process), prostate cancer (cancer type) and platinum-based anti-cancer drugs including carboplatin, cisplatin, and oxaliplatin (anti-cancer drugs). The detailed information extracted from the literature includes basic information about the articles (e.g., PubMed ID, authors, journal name, publication year), information about the samples (type, study/reference, prognosis factor), and the proteomics workflow (Subcellular fractionation, protein, and peptide separation, mass spectrometry, quantification). Useful annotations such as hyperlinks to UniProt and PubMed were included. In addition, many filtering options were established as well as export functions. The database is freely available at http://cancerproteomics.uio.no. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Liu, Xing; Xu, Yanli; Meng, Qian

Colorectal cancer (CRC) is one of the most common types of malignant tumor worldwide. Currently, although many researchers have been devoting themselves in CRC studies, the process of locating biomarkers for CRC early diagnosis and prognostic is still very slow. Using a centrifugal proteomic reactor-based proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling, 2620 protein groups were quantified between cancer mucosa and adjacent normal colorectal mucosa. Of which, 403 protein groups were differentially expressed with statistic significance between cancer and normal tissues, including 195 up-regulated and 208 down-regulated proteins in cancer tissues. Three proteins (SOD3, PRELP andmore » NGAL) were selected for further Western blot validation. And the resulting Western blot experimental results were consistent with the quantitative proteomic data. SOD3 and PRELP are down-regulated in CRC mucosa comparing to adjacent normal tissue, while NGAL is up-regulated in CRC mucosa. In conclusion, the centrifugal proteomic reactor-based label-free quantitative proteomic approach provides a highly sensitive and powerful tool for analyzing minute protein sample from tiny colorectal biopsies, which may facilitate CRC biomarkers discovery for diagnoses and prognoses. -- Highlights: •Minute amount of colonic biopsies by endoscopy is suitable for proteomic analysis. •Centrifugal proteomic reactor can be used for processing tiny clinic biopsy sample. •SOD3 and PRELP are down-regulated in CRC, while NGAL is up-regulated in CRC.« less

Bioinformatics strategies in life sciences: from data processing and data warehousing to biological knowledge extraction.

PubMed

Thiele, Herbert; Glandorf, Jörg; Hufnagel, Peter

2010-05-27

With the large variety of Proteomics workflows, as well as the large variety of instruments and data-analysis software available, researchers today face major challenges validating and comparing their Proteomics data. Here we present a new generation of the ProteinScape bioinformatics platform, now enabling researchers to manage Proteomics data from the generation and data warehousing to a central data repository with a strong focus on the improved accuracy, reproducibility and comparability demanded by many researchers in the field. It addresses scientists; current needs in proteomics identification, quantification and validation. But producing large protein lists is not the end point in Proteomics, where one ultimately aims to answer specific questions about the biological condition or disease model of the analyzed sample. In this context, a new tool has been developed at the Spanish Centro Nacional de Biotecnologia Proteomics Facility termed PIKE (Protein information and Knowledge Extractor) that allows researchers to control, filter and access specific information from genomics and proteomic databases, to understand the role and relationships of the proteins identified in the experiments. Additionally, an EU funded project, ProDac, has coordinated systematic data collection in public standards-compliant repositories like PRIDE. This will cover all aspects from generating MS data in the laboratory, assembling the whole annotation information and storing it together with identifications in a standardised format.

Chimeric plastid proteome in the Florida "red tide" dinoflagellate Karenia brevis.

PubMed

Nosenko, Tetyana; Lidie, Kristy L; Van Dolah, Frances M; Lindquist, Erika; Cheng, Jan-Fang; Bhattacharya, Debashish

2006-11-01

Current understanding of the plastid proteome comes almost exclusively from studies of plants and red algae. The proteome in these taxa has a relatively simple origin via integration of proteins from a single cyanobacterial primary endosymbiont and the host. However, the most successful algae in marine environments are the chlorophyll c-containing chromalveolates such as diatoms and dinoflagellates that contain a plastid of red algal origin derived via secondary or tertiary endosymbiosis. Virtually nothing is known about the plastid proteome in these taxa. We analyzed expressed sequence tag data from the toxic "Florida red tide" dinoflagellate Karenia brevis that has undergone a tertiary plastid endosymbiosis. Comparative analyses identified 30 nuclear-encoded plastid-targeted proteins in this chromalveolate that originated via endosymbiotic or horizontal gene transfer (HGT) from multiple different sources. We identify a fundamental divide between plant/red algal and chromalveolate plastid proteomes that reflects a history of mixotrophy in the latter group resulting in a highly chimeric proteome. Loss of phagocytosis in the "red" and "green" clades effectively froze their proteomes, whereas chromalveolate lineages retain the ability to engulf prey allowing them to continually recruit new, potentially adaptive genes through subsequent endosymbioses and HGT. One of these genes is an electron transfer protein (plastocyanin) of green algal origin in K. brevis that likely allows this species to thrive under conditions of iron depletion.

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

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

Multiplexed immunofluorescence delineates proteomic cancer cell states associated with metabolism

PubMed Central

Sood, Anup; Miller, Alexandra M.; Brogi, Edi; Sui, Yunxia; Armenia, Joshua; McDonough, Elizabeth; Santamaria-Pang, Alberto; Stamper, Aleksandra; Campos, Carl; Pang, Zhengyu; Li, Qing; Port, Elisa; Graeber, Thomas G.; Schultz, Nikolaus; Ginty, Fiona; Larson, Steven M.

2016-01-01

The phenotypic diversity of cancer results from genetic and nongenetic factors. Most studies of cancer heterogeneity have focused on DNA alterations, as technologies for proteomic measurements in clinical specimen are currently less advanced. Here, we used a multiplexed immunofluorescence staining platform to measure the expression of 27 proteins at the single-cell level in formalin-fixed and paraffin-embedded samples from treatment-naive stage II/III human breast cancer. Unsupervised clustering of protein expression data from 638,577 tumor cells in 26 breast cancers identified 8 clusters of protein coexpression. In about one-third of breast cancers, over 95% of all neoplastic cells expressed a single protein coexpression cluster. The remaining tumors harbored tumor cells representing multiple protein coexpression clusters, either in a regional distribution or intermingled throughout the tumor. Tumor uptake of the radiotracer 18F-fluorodeoxyglucose was associated with protein expression clusters characterized by hormone receptor loss, PTEN alteration, and HER2 gene amplification. Our study demonstrates an approach to generate cellular heterogeneity metrics in routinely collected solid tumor specimens and integrate them with in vivo cancer phenotypes. PMID:27182557

Targeted proteomics coming of age - SRM, PRM and DIA performance evaluated from a core facility perspective.

PubMed

Kockmann, Tobias; Trachsel, Christian; Panse, Christian; Wahlander, Asa; Selevsek, Nathalie; Grossmann, Jonas; Wolski, Witold E; Schlapbach, Ralph

2016-08-01

Quantitative mass spectrometry is a rapidly evolving methodology applied in a large number of omics-type research projects. During the past years, new designs of mass spectrometers have been developed and launched as commercial systems while in parallel new data acquisition schemes and data analysis paradigms have been introduced. Core facilities provide access to such technologies, but also actively support the researchers in finding and applying the best-suited analytical approach. In order to implement a solid fundament for this decision making process, core facilities need to constantly compare and benchmark the various approaches. In this article we compare the quantitative accuracy and precision of current state of the art targeted proteomics approaches single reaction monitoring (SRM), parallel reaction monitoring (PRM) and data independent acquisition (DIA) across multiple liquid chromatography mass spectrometry (LC-MS) platforms, using a readily available commercial standard sample. All workflows are able to reproducibly generate accurate quantitative data. However, SRM and PRM workflows show higher accuracy and precision compared to DIA approaches, especially when analyzing low concentrated analytes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The HUPO proteomics standards initiative--easing communication and minimizing data loss in a changing world.

PubMed

Orchard, Sandra; Hermjakob, Henning

2008-03-01

The amount of data currently being generated by proteomics laboratories around the world is increasing exponentially, making it ever more critical that scientists are able to exchange, compare and retrieve datasets when re-evaluation of their original conclusions becomes important. Only a fraction of this data is published in the literature and important information is being lost every day as data formats become obsolete. The Human Proteome Organisation Proteomics Standards Initiative (HUPO-PSI) was tasked with the creation of data standards and interchange formats to allow both the exchange and storage of such data irrespective of the hardware and software from which it was generated. This article will provide an update on the work of this group, the creation and implementation of these standards and the standards-compliant data repositories being established as result of their efforts.

From the genome sequence to the protein inventory of Bacillus subtilis.

PubMed

Becher, Dörte; Büttner, Knut; Moche, Martin; Hessling, Bernd; Hecker, Michael

2011-08-01

Owing to the low number of proteins necessary to render a bacterial cell viable, bacteria are extremely attractive model systems to understand how the genome sequence is translated into actual life processes. One of the most intensively investigated model organisms is Bacillus subtilis. It has attracted world-wide research interest, addressing cell differentiation and adaptation on a molecular scale as well as biotechnological production processes. Meanwhile, we are looking back on more than 25 years of B. subtilis proteomics. A wide range of methods have been developed during this period for the large-scale qualitative and quantitative proteome analysis. Currently, it is possible to identify and quantify more than 50% of the predicted proteome in different cellular subfractions. In this review, we summarize the development of B. subtilis proteomics during the past 25 years. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

P-MartCancer-Interactive Online Software to Enable Analysis of Shotgun Cancer Proteomic Datasets.

PubMed

Webb-Robertson, Bobbie-Jo M; Bramer, Lisa M; Jensen, Jeffrey L; Kobold, Markus A; Stratton, Kelly G; White, Amanda M; Rodland, Karin D

2017-11-01

P-MartCancer is an interactive web-based software environment that enables statistical analyses of peptide or protein data, quantitated from mass spectrometry-based global proteomics experiments, without requiring in-depth knowledge of statistical programming. P-MartCancer offers a series of statistical modules associated with quality assessment, peptide and protein statistics, protein quantification, and exploratory data analyses driven by the user via customized workflows and interactive visualization. Currently, P-MartCancer offers access and the capability to analyze multiple cancer proteomic datasets generated through the Clinical Proteomics Tumor Analysis Consortium at the peptide, gene, and protein levels. P-MartCancer is deployed as a web service (https://pmart.labworks.org/cptac.html), alternatively available via Docker Hub (https://hub.docker.com/r/pnnl/pmart-web/). Cancer Res; 77(21); e47-50. ©2017 AACR . ©2017 American Association for Cancer Research.

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.

Video Release: 47th Vice President of the United States Joseph R. Biden Jr. Speech at HUPO2017 Global Leadership Gala | Office of Cancer Clinical Proteomics Research

Cancer.gov

The Human Proteome Organization (HUPO) has released a video of the keynote speech given by the 47th Vice President of the United States of America Joseph R. Biden Jr. at the HUPO2017 Global Leadership Gala. Under the gala theme “International Cooperation in the Fight Against Cancer,” Biden recognized cancer as a collection of related diseases, the importance of data sharing and harmonization, and the need for collaboration across scientific disciplines as inflection points in cancer research.

Recent advances in stable isotope labeling based techniques for proteome relative quantification.

PubMed

Zhou, Yuan; Shan, Yichu; Zhang, Lihua; Zhang, Yukui

2014-10-24

The large scale relative quantification of all proteins expressed in biological samples under different states is of great importance for discovering proteins with important biological functions, as well as screening disease related biomarkers and drug targets. Therefore, the accurate quantification of proteins at proteome level has become one of the key issues in protein science. Herein, the recent advances in stable isotope labeling based techniques for proteome relative quantification were reviewed, from the aspects of metabolic labeling, chemical labeling and enzyme-catalyzed labeling. Furthermore, the future research direction in this field was prospected. Copyright © 2014 Elsevier B.V. All rights reserved.

Proteomics of ovarian cancer: functional insights and clinical applications

DOE PAGES

Elzek, Mohamed A.; Rodland, Karin D.

2015-03-04

In the past decade, there has been an increasing interest in applying proteomics to assist in understanding the pathogenesis of ovarian cancer, elucidating the mechanism of drug resistance, and in the development of biomarkers for early detection of ovarian cancer. Although ovarian cancer is a spectrum of different diseases, the strategies for diagnosis and treatment with surgery and adjuvant therapy are similar across ovarian cancer types, increasing the general applicability of discoveries made through proteomics research. While proteomic experiments face many difficulties which slow the pace of clinical applications, recent advances in proteomic technology contribute significantly to the identification ofmore » aberrant proteins and networks which can serve as targets for biomarker development and individualized therapies. This review provides a summary of the literature on proteomics’ contributions to ovarian cancer research and highlights the current issues, future directions, and challenges. In conclusion, we propose that protein-level characterization of primary lesion in ovarian cancer can decipher the mystery of this disease, improve diagnostic tools, and lead to more effective screening programs.« less

Literature Mining of Pathogenesis-Related Proteins in Human Pathogens for Database Annotation

DTIC Science & Technology

2009-10-01

person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control...submission and for literature mining result display with automatically tagged abstracts. I. Literature data sets for machine learning algorithm training...mass spectrometry) proteomics data from Burkholderia strains. • Task1 ( M13 -15): Preliminary analysis of the Burkholderia proteomic space

Proteomics of survival structures of fungal pathogens.

PubMed

Loginov, Dmitry; Šebela, Marek

2016-09-25

Fungal pathogens are causal agents of numerous human, animal, and plant diseases. They employ various infection modes to overcome host defense systems. Infection mechanisms of different fungi have been subjected to many comprehensive studies. These investigations have been facilitated by the development of various '-omics' techniques, and proteomics has one of the leading roles in this regard. Fungal conidia and sclerotia could be considered the most important structures for pathogenesis as their germination is one of the first steps towards a host infection. They represent interesting objects for proteomic studies because of the presence of unique proteins with unexplored biotechnological potential required for pathogen viability, development and the subsequent host infection. Proteomic peculiarities of survival structures of different fungi, including those of biotechnological significance (e.g., Asperillus fumigatus, A. nidulans, Metarhizium anisopliae), in a dormant state, as well as changes in the protein production during early stages of fungal development are the subjects of the present review. We focused on biological aspects of proteomic studies of fungal survival structures rather than on an evaluation of proteomic approaches. For that reason, proteins that have been identified in this context are discussed from the point of view of their involvement in different biological processes and possible functions assigned to them. This is the first review paper summarizing recent advances in proteomics of fungal survival structures. Copyright © 2016 Elsevier B.V. All rights reserved.

pGlyco 2.0 enables precision N-glycoproteomics with comprehensive quality control and one-step mass spectrometry for intact glycopeptide identification.

PubMed

Liu, Ming-Qi; Zeng, Wen-Feng; Fang, Pan; Cao, Wei-Qian; Liu, Chao; Yan, Guo-Quan; Zhang, Yang; Peng, Chao; Wu, Jian-Qiang; Zhang, Xiao-Jin; Tu, Hui-Jun; Chi, Hao; Sun, Rui-Xiang; Cao, Yong; Dong, Meng-Qiu; Jiang, Bi-Yun; Huang, Jiang-Ming; Shen, Hua-Li; Wong, Catherine C L; He, Si-Min; Yang, Peng-Yuan

2017-09-05

The precise and large-scale identification of intact glycopeptides is a critical step in glycoproteomics. Owing to the complexity of glycosylation, the current overall throughput, data quality and accessibility of intact glycopeptide identification lack behind those in routine proteomic analyses. Here, we propose a workflow for the precise high-throughput identification of intact N-glycopeptides at the proteome scale using stepped-energy fragmentation and a dedicated search engine. pGlyco 2.0 conducts comprehensive quality control including false discovery rate evaluation at all three levels of matches to glycans, peptides and glycopeptides, improving the current level of accuracy of intact glycopeptide identification. The N-glycoproteome of samples metabolically labeled with 15 N/ 13 C were analyzed quantitatively and utilized to validate the glycopeptide identification, which could be used as a novel benchmark pipeline to compare different search engines. Finally, we report a large-scale glycoproteome dataset consisting of 10,009 distinct site-specific N-glycans on 1988 glycosylation sites from 955 glycoproteins in five mouse tissues.Protein glycosylation is a heterogeneous post-translational modification that generates greater proteomic diversity that is difficult to analyze. Here the authors describe pGlyco 2.0, a workflow for the precise one step identification of intact N-glycopeptides at the proteome scale.

Time-resolved Global and Chromatin Proteomics during Herpes Simplex Virus Type 1 (HSV-1) Infection.

PubMed

Kulej, Katarzyna; Avgousti, Daphne C; Sidoli, Simone; Herrmann, Christin; Della Fera, Ashley N; Kim, Eui Tae; Garcia, Benjamin A; Weitzman, Matthew D

2017-04-01

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. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteomics and metabolomics characterizing the pathophysiology of adaptive reactions to the metabolic challenges during the transition from late pregnancy to early lactation in dairy cows.

PubMed

Ceciliani, Fabrizio; Lecchi, Cristina; Urh, Christiane; Sauerwein, Helga

2018-04-30

The transition from late pregnancy to early lactation is a critical period in a dairy cow's life due to the rapidly increasing drain of nutrients from the maternal organism towards the foetus and into colostrum and milk. In order to cope with the challenges of parturition and lactation, comprehensive adaptive reactions comprising the endocrine and the immune system need to be accomplished. There is high variation in this coping ability and both metabolic and infectious diseases, summarized as "production diseases", such as hypocalcaemia (milk fever), fatty liver syndrome, laminitis and ketosis, may occur and impact welfare, productive lifespan and economic outcomes. Proteomics and metabolomics have emerged as valuable techniques to characterize proteins and metabolite assets from tissue and biological fluids, such as milk, blood and urine. In this review we provide an overview on metabolic status and physiological changes during the transition period and the related production diseases in dairy cows, and summarize the state of art on proteomics and metabolomics of biological fluids and tissues involved in metabolic stress during the peripartum period. We also provide a current and prospective view of the application of the recent achievements generated by omics for biomarker discovery and their potential in diagnosis. For high-yielding dairy cows there are several "occupational diseases" that occur mainly during the metabolic challenges related to the transition from pregnancy to lactation. Such diseases and their sequelae form a major concern for dairy production, and often lead to early culling of animals. Beside the economical perspective, metabolic stress may severely influence animal welfare. There is a multitude of studies about the metabolic backgrounds of such so called production diseases like ketosis, fatty liver, or hypocalcaemia, although the investigations aiming to assess the complexity of the pathophysiological reactions are largely focused on gene expression, i.e. transcriptomics. For extending the knowledge towards the proteome and the metabolome, the respective technologies are of increasing importance and can provide an overall view of how dairy cows react to metabolic stress, which is needed for an in-depth understanding of the molecular mechanisms of the related diseases. We herein review the current findings from studies applying proteomics and metabolomics to transition-related diseases, including fatty liver, ketosis, endometritis, hypocalcaemia and laminitis. For each disease, a brief overview of the up to date knowledge about its pathogenesis is provided, followed by an insight into the most recent achievements on the proteome and metabolome of tissues and biological fluids, such as blood serum and urine, highlighting potential biomarkers. We believe that this review would help readers to be become more familiar with the recent progresses of molecular background of transition-related diseases thus encouraging research in this field. Copyright © 2017 Elsevier B.V. All rights reserved.

Global Cell Proteome Profiling, Phospho-signaling and Quantitative 
Proteomics for Identification of New Biomarkers in Acute Myeloid 
Leukemia Patients

PubMed Central

Aasebø, Elise; Forthun, Rakel B.; Berven, Frode; Selheim, Frode; Hernandez-Valladares, Maria

2016-01-01

The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applications in AML diagnosis and prognosis, treatment and the selection for bone marrow transplant requires substantial comparative analyses of the proteomes from AML patients. In the past years, several studies have suggested some biomarkers for AML diagnosis or AML classification using methods for sample preparation with low proteome coverage and low resolution mass spectrometers. However, most of the studies did not follow up, confirm or validate their candidates with more patient samples. Current proteomics methods, new high resolution and fast mass spectrometers allow the identification and quantification of several thousands of proteins obtained from few tens of μg of AML cell lysate. Enrichment methods for posttranslational modifications (PTM), such as phosphorylation, can isolate several thousands of site-specific phosphorylated peptides from AML patient samples, which subsequently can be quantified with high confidence in new mass spectrometers. While recent reports aiming to propose proteomic or phosphoproteomic biomarkers on the studied AML patient samples have taken advantage of the technological progress, the access to large cohorts of AML patients to sample from and the availability of appropriate control samples still remain challenging. PMID:26306748

Development of proteome-wide binding reagents for research and diagnostics.

PubMed

Taussig, Michael J; Schmidt, Ronny; Cook, Elizabeth A; Stoevesandt, Oda

2013-12-01

Alongside MS, antibodies and other specific protein-binding molecules have a special place in proteomics as affinity reagents in a toolbox of applications for determining protein location, quantitative distribution and function (affinity proteomics). The realisation that the range of research antibodies available, while apparently vast is nevertheless still very incomplete and frequently of uncertain quality, has stimulated projects with an objective of raising comprehensive, proteome-wide sets of protein binders. With progress in automation and throughput, a remarkable number of recent publications refer to the practical possibility of selecting binders to every protein encoded in the genome. Here we review the requirements of a pipeline of production of protein binders for the human proteome, including target prioritisation, antigen design, 'next generation' methods, databases and the approaches taken by ongoing projects in Europe and the USA. While the task of generating affinity reagents for all human proteins is complex and demanding, the benefits of well-characterised and quality-controlled pan-proteome binder resources for biomedical research, industry and life sciences in general would be enormous and justify the effort. Given the technical, personnel and financial resources needed to fulfil this aim, expansion of current efforts may best be addressed through large-scale international collaboration. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A decade of Central and Eastern European Proteomic Conference (CEEPC): Credibility, cohesion and vision for the next decade.

PubMed

Gadher, Suresh Jivan; Kovarova, Hana

2017-02-05

The Central and Eastern European Proteomic Conference (CEEPC), has reached a special milestone as it celebrates its 10th anniversary. Today, an expansive network of proteomics in Central and Eastern Europe stands established to facilitate scientific interactions and collaborations in and around Central and Eastern Europe, as well as with international research institutions worldwide. Currently, when many conferences are struggling to attract participants, CEEPC is thriving in its status and stature as well as expanding by attracting newer member countries. CEEPC's success is driven by mutual respect between scientists sharing interest in proteomics and its applications in multidisciplinary research areas related to biological systems. This effort when interwoven with exciting ambience steeped with culture, and tradition is also a reason why participants enjoy it. CEEPC's careful balance between excellence and cohesion holds the key to its success. It is evident that CEEPC is ready for the next decade of excitement and expectations of multifaceted proteomics in Central and Eastern Europe. Additionally, in the era of emerging personalized medicine where treatment selection for each patient is becoming individualized, CEEPC and proteomics is expected to play a significant role moving forward for the benefit of mankind. Copyright © 2016 Elsevier B.V. All rights reserved.

Integration of cardiac proteome biology and medicine by a specialized knowledgebase.

PubMed

Zong, Nobel C; Li, Haomin; Li, Hua; Lam, Maggie P Y; Jimenez, Rafael C; Kim, Christina S; Deng, Ning; Kim, Allen K; Choi, Jeong Ho; Zelaya, Ivette; Liem, David; Meyer, David; Odeberg, Jacob; Fang, Caiyun; Lu, Hao-Jie; Xu, Tao; Weiss, James; Duan, Huilong; Uhlen, Mathias; Yates, John R; Apweiler, Rolf; Ge, Junbo; Hermjakob, Henning; Ping, Peipei

2013-10-12

Omics sciences enable a systems-level perspective in characterizing cardiovascular biology. Integration of diverse proteomics data via a computational strategy will catalyze the assembly of contextualized knowledge, foster discoveries through multidisciplinary investigations, and minimize unnecessary redundancy in research efforts. The goal of this project is to develop a consolidated cardiac proteome knowledgebase with novel bioinformatics pipeline and Web portals, thereby serving as a new resource to advance cardiovascular biology and medicine. We created Cardiac Organellar Protein Atlas Knowledgebase (COPaKB; www.HeartProteome.org), a centralized platform of high-quality cardiac proteomic data, bioinformatics tools, and relevant cardiovascular phenotypes. Currently, COPaKB features 8 organellar modules, comprising 4203 LC-MS/MS experiments from human, mouse, drosophila, and Caenorhabditis elegans, as well as expression images of 10,924 proteins in human myocardium. In addition, the Java-coded bioinformatics tools provided by COPaKB enable cardiovascular investigators in all disciplines to retrieve and analyze pertinent organellar protein properties of interest. COPaKB provides an innovative and interactive resource that connects research interests with the new biological discoveries in protein sciences. With an array of intuitive tools in this unified Web server, nonproteomics investigators can conveniently collaborate with proteomics specialists to dissect the molecular signatures of cardiovascular phenotypes.

Microbial Interactions in Plants: Perspectives and Applications of Proteomics.

PubMed

Imam, Jahangir; Shukla, Pratyoosh; Mandal, Nimai Prasad; Variar, Mukund

2017-01-01

The structure and function of proteins involved in plant-microbe interactions is investigated through large-scale proteomics technology in a complex biological sample. Since the whole genome sequences are now available for several plant species and microbes, proteomics study has become easier, accurate and huge amount of data can be generated and analyzed during plant-microbe interactions. Proteomics approaches are highly important and relevant in many studies and showed that only genomics approaches are not sufficient enough as much significant information are lost as the proteins and not the genes coding them are final product that is responsible for the observed phenotype. Novel approaches in proteomics are developing continuously enabling the study of the various aspects in arrangements and configuration of proteins and its functions. Its application is becoming more common and frequently used in plant-microbe interactions with the advancement in new technologies. They are more used for the portrayal of cell and extracellular destructiveness and pathogenicity variables delivered by pathogens. This distinguishes the protein level adjustments in host plants when infected with pathogens and advantageous partners. This review provides a brief overview of different proteomics technology which is currently available followed by their exploitation to study the plant-microbe interaction. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides.

PubMed

Tsirigos, Konstantinos D; Peters, Christoph; Shu, Nanjiang; Käll, Lukas; Elofsson, Arne

2015-07-01

TOPCONS (http://topcons.net/) is a widely used web server for consensus prediction of membrane protein topology. We hereby present a major update to the server, with some substantial improvements, including the following: (i) TOPCONS can now efficiently separate signal peptides from transmembrane regions. (ii) The server can now differentiate more successfully between globular and membrane proteins. (iii) The server now is even slightly faster, although a much larger database is used to generate the multiple sequence alignments. For most proteins, the final prediction is produced in a matter of seconds. (iv) The user-friendly interface is retained, with the additional feature of submitting batch files and accessing the server programmatically using standard interfaces, making it thus ideal for proteome-wide analyses. Indicatively, the user can now scan the entire human proteome in a few days. (v) For proteins with homology to a known 3D structure, the homology-inferred topology is also displayed. (vi) Finally, the combination of methods currently implemented achieves an overall increase in performance by 4% as compared to the currently available best-scoring methods and TOPCONS is the only method that can identify signal peptides and still maintain a state-of-the-art performance in topology predictions. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Formaldehyde cross-linking and structural proteomics: Bridging the gap.

PubMed

Srinivasa, Savita; Ding, Xuan; Kast, Juergen

2015-11-01

Proteins are dynamic entities constantly moving and altering their structures based on their functions and interactions inside and outside the cell. Formaldehyde cross-linking combined with mass spectrometry can accurately capture interactions of these rapidly changing biomolecules while maintaining their physiological surroundings. Even with its numerous established uses in biology and compatibility with mass spectrometry, formaldehyde has not yet been applied in structural proteomics. However, formaldehyde cross-linking is moving toward analyzing tertiary structure, which conventional cross-linkers have already accomplished. The purpose of this review is to describe the potential of formaldehyde cross-linking in structural proteomics by highlighting its applications, characteristics and current status in the field. Copyright © 2015 Elsevier Inc. All rights reserved.

Functional protease profiling for diagnosis of malignant disease.

PubMed

Findeisen, Peter; Neumaier, Michael

2012-01-01

Clinical proteomic profiling by mass spectrometry (MS) aims at uncovering specific alterations within mass profiles of clinical specimens that are of diagnostic value for the detection and classification of various diseases including cancer. However, despite substantial progress in the field, the clinical proteomic profiling approaches have not matured into routine diagnostic applications so far. Their limitations are mainly related to high-abundance proteins and their complex processing by a multitude of endogenous proteases thus making rigorous standardization difficult. MS is biased towards the detection of low-molecular-weight peptides. Specifically, in serum specimens, the particular fragments of proteolytically degraded proteins are amenable to MS analysis. Proteases are known to be involved in tumour progression and tumour-specific proteases are released into the blood stream presumably as a result of invasive progression and metastasis. Thus, the determination of protease activity in clinical specimens from patients with malignant disease can offer diagnostic and also therapeutic options. The identification of specific substrates for tumour proteases in complex biological samples is challenging, but proteomic screens for proteases/substrate interactions are currently experiencing impressive progress. Such proteomic screens include peptide-based libraries, differential isotope labelling in combination with MS, quantitative degradomic analysis of proteolytically generated neo-N-termini, monitoring the degradation of exogenous reporter peptides with MS, and activity-based protein profiling. In the present article, we summarize and discuss the current status of proteomic techniques to identify tumour-specific protease-substrate interactions for functional protease profiling. Thereby, we focus on the potential diagnostic use of the respective approaches. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preparation of the low molecular weight serum proteome for mass spectrometry analysis.

PubMed

Waybright, Timothy J; Chan, King C; Veenstra, Timothy D; Xiao, Zhen

2013-01-01

The discovery of viable biomarkers or indicators of disease states is complicated by the inherent complexity of the chosen biological specimen. Every sample, whether it is serum, plasma, urine, tissue, cells, or a host of others, contains thousands of large and small components, each interacting in multiple ways. The need to concentrate on a group of these components to narrow the focus on a potential biomarker candidate becomes, out of necessity, a priority, especially in the search for immune-related low molecular weight serum biomarkers. One such method in the field of proteomics is to divide the sample proteome into groups based on the size of the protein, analyze each group, and mine the data for statistically significant items. This chapter details a portion of this method, concentrating on a method for fractionating and analyzing the low molecular weight proteome of human serum.

Differential proteome analysis of diabetes mellitus type 2 and its pathophysiological complications.

PubMed

Sohail, Waleed; Majeed, Fatimah; Afroz, Amber

2018-06-11

The prevalence of Diabetes Mellitus Type 2 (DM 2) is increasing every passing year due to some global changes in lifestyles of people. The exact underlying mechanisms of the progression of this disease are not yet known. However recent advances in the combined omics more particularly in proteomics and genomics have opened a gateway towards the understanding of predetermined genetic factors, progression, complications and treatment of this disease. Here we shall review the recent advances in proteomics that have led to an early and better diagnostic approaches in controlling DM 2 more importantly the comparison of structural and functional protein biomarkers that are modified in the diseased state. By applying these advanced and promising proteomic strategies with bioinformatics applications and bio-statistical tools the prevalence of DM 2 and its associated disorders i-e nephropathy and retinopathy are expected to be controlled. Copyright © 2018 Diabetes India. Published by Elsevier Ltd. All rights reserved.

Redox Proteomics of Protein-bound Methionine Oxidation*

PubMed Central

Ghesquière, Bart; Jonckheere, Veronique; Colaert, Niklaas; Van Durme, Joost; Timmerman, Evy; Goethals, Marc; Schymkowitz, Joost; Rousseau, Frederic; Vandekerckhove, Joël; Gevaert, Kris

2011-01-01

We here present a new method to measure the degree of protein-bound methionine sulfoxide formation at a proteome-wide scale. In human Jurkat cells that were stressed with hydrogen peroxide, over 2000 oxidation-sensitive methionines in more than 1600 different proteins were mapped and their extent of oxidation was quantified. Meta-analysis of the sequences surrounding the oxidized methionine residues revealed a high preference for neighboring polar residues. Using synthetic methionine sulfoxide containing peptides designed according to the observed sequence preferences in the oxidized Jurkat proteome, we discovered that the substrate specificity of the cellular methionine sulfoxide reductases is a major determinant for the steady-state of methionine oxidation. This was supported by a structural modeling of the MsrA catalytic center. Finally, we applied our method onto a serum proteome from a mouse sepsis model and identified 35 in vivo methionine oxidation events in 27 different proteins. PMID:21406390

Standardized protocols for quality control of MRM-based plasma proteomic workflows.

PubMed

Percy, Andrew J; Chambers, Andrew G; Smith, Derek S; Borchers, Christoph H

2013-01-04

Mass spectrometry (MS)-based proteomics is rapidly emerging as a viable technology for the identification and quantitation of biological samples, such as human plasma--the most complex yet commonly employed biofluid in clinical analyses. The transition from a qualitative to quantitative science is required if proteomics is going to successfully make the transition to a clinically useful technique. MS, however, has been criticized for a lack of reproducibility and interlaboratory transferability. Currently, the MS and plasma proteomics communities lack standardized protocols and reagents to ensure that high-quality quantitative data can be accurately and precisely reproduced by laboratories across the world using different MS technologies. Toward addressing this issue, we have developed standard protocols for multiple reaction monitoring (MRM)-based assays with customized isotopically labeled internal standards for quality control of the sample preparation workflow and the MS platform in quantitative plasma proteomic analyses. The development of reference standards and their application to a single MS platform is discussed herein, along with the results from intralaboratory tests. The tests highlighted the importance of the reference standards in assessing the efficiency and reproducibility of the entire bottom-up proteomic workflow and revealed errors related to the sample preparation and performance quality and deficits of the MS and LC systems. Such evaluations are necessary if MRM-based quantitative plasma proteomics is to be used in verifying and validating putative disease biomarkers across different research laboratories and eventually in clinical laboratories.

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

Neural Stem Cells (NSCs) and Proteomics.

PubMed

Shoemaker, Lorelei D; Kornblum, Harley I

2016-02-01

Neural stem cells (NSCs) can self-renew and give rise to the major cell types of the CNS. Studies of NSCs include the investigation of primary, CNS-derived cells as well as animal and human embryonic stem cell (ESC)-derived and induced pluripotent stem cell (iPSC)-derived sources. NSCs provide a means with which to study normal neural development, neurodegeneration, and neurological disease and are clinically relevant sources for cellular repair to the damaged and diseased CNS. Proteomics studies of NSCs have the potential to delineate molecules and pathways critical for NSC biology and the means by which NSCs can participate in neural repair. In this review, we provide a background to NSC biology, including the means to obtain them and the caveats to these processes. We then focus on advances in the proteomic interrogation of NSCs. This includes the analysis of posttranslational modifications (PTMs); approaches to analyzing different proteomic compartments, such the secretome; as well as approaches to analyzing temporal differences in the proteome to elucidate mechanisms of differentiation. We also discuss some of the methods that will undoubtedly be useful in the investigation of NSCs but which have not yet been applied to the field. While many proteomics studies of NSCs have largely catalogued the proteome or posttranslational modifications of specific cellular states, without delving into specific functions, some have led to understandings of functional processes or identified markers that could not have been identified via other means. Many challenges remain in the field, including the precise identification and standardization of NSCs used for proteomic analyses, as well as how to translate fundamental proteomics studies to functional biology. The next level of investigation will require interdisciplinary approaches, combining the skills of those interested in the biochemistry of proteomics with those interested in modulating NSC function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Development of Best practices document for Peptide Standards | Office of Cancer Clinical Proteomics Research

Cancer.gov

The Assay Development Working Group (ADWG) of the CPTAC Program is currently drafting a document to propose best practices for generation, quantification, storage, and handling of peptide standards used for mass spectrometry-based assays, as well as interpretation of quantitative proteomic data based on peptide standards. The ADWG is seeking input from commercial entities that provide peptide standards for mass spectrometry-based assays or that perform amino acid analysis.

Differential Cysteine Labeling and Global Label-Free Proteomics Reveals an Altered Metabolic State in Skeletal Muscle Aging

PubMed Central

2014-01-01

The molecular mechanisms underlying skeletal muscle aging and associated sarcopenia have been linked to an altered oxidative status of redox-sensitive proteins. Reactive oxygen and reactive nitrogen species (ROS/RNS) generated by contracting skeletal muscle are necessary for optimal protein function, signaling, and adaptation. To investigate the redox proteome of aging gastrocnemius muscles from adult and old male mice, we developed a label-free quantitative proteomic approach that includes a differential cysteine labeling step. The approach allows simultaneous identification of up- and downregulated proteins between samples in addition to the identification and relative quantification of the reversible oxidation state of susceptible redox cysteine residues. Results from muscles of adult and old mice indicate significant changes in the content of chaperone, glucose metabolism, and cytoskeletal regulatory proteins, including Protein DJ-1, cAMP-dependent protein kinase type II, 78 kDa glucose regulated protein, and a reduction in the number of redox-responsive proteins identified in muscle of old mice. Results demonstrate skeletal muscle aging causes a reduction in redox-sensitive proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response. Data is available via ProteomeXchange with identifier PXD001054. PMID:25181601

A set of ligation-independent in vitro translation vectors for eukaryotic protein production.

PubMed

Bardóczy, Viola; Géczi, Viktória; Sawasaki, Tatsuya; Endo, Yaeta; Mészáros, Tamás

2008-03-27

The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags. We designed four ligation independent cloning (LIC) vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein. Four newly designed in vitro translation vectors have been constructed which allow fast and parallel cloning and protein purification, thus representing useful molecular tools for high-throughput production of eukaryotic proteins.

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.

Molecular diagnostics in medical microbiology: yesterday, today and tomorrow.

PubMed

van Belkum, Alex

2003-10-01

Clinical microbiology is clearly on the move, and various new diagnostic technologies have been introduced into laboratory practice over the past few decades. However, Henri D Isenberg recently stated that molecular biology techniques promised to revolutionise the diagnosis of infectious disease, but that, to date, this promise is still in its infancy. Molecular diagnostics have now surpassed these early stages and have definitely reached puberty. Currently, a second generation of automated molecular approaches is already within the microbiologists' reach. Quantitative amplification tests in combination with genomics, transcriptomics, proteomics and related methodologies will pave the way to further enhancement of innovative microbial detection and identification.

Metabolic changes associated with the long winter fast dominate the liver proteome in 13-lined ground squirrels

PubMed Central

Hindle, Allyson G.; Grabek, Katharine R.; Epperson, L. Elaine; Karimpour-Fard, Anis

2014-01-01

Small-bodied hibernators partition the year between active homeothermy and hibernating heterothermy accompanied by fasting. To define molecular events underlying hibernation that are both dependent and independent of fasting, we analyzed the liver proteome among two active and four hibernation states in 13-lined ground squirrels. We also examined fall animals transitioning between fed homeothermy and fasting heterothermy. Significantly enriched pathways differing between activity and hibernation were biased toward metabolic enzymes, concordant with the fuel shifts accompanying fasting physiology. Although metabolic reprogramming to support fasting dominated these data, arousing (rewarming) animals had the most distinct proteome among the hibernation states. Instead of a dominant metabolic enzyme signature, torpor-arousal cycles featured differences in plasma proteins and intracellular membrane traffic and its regulation. Phosphorylated NSFL1C, a membrane regulator, exhibited this torpor-arousal cycle pattern; its role in autophagosome formation may promote utilization of local substrates upon metabolic reactivation in arousal. Fall animals transitioning to hibernation lagged in their proteomic adjustment, indicating that the liver is more responsive than preparatory to the metabolic reprogramming of hibernation. Specifically, torpor use had little impact on the fall liver proteome, consistent with a dominant role of nutritional status. In contrast to our prediction of reprogramming the transition between activity and hibernation by gene expression and then within-hibernation transitions by posttranslational modification (PTM), we found extremely limited evidence of reversible PTMs within torpor-arousal cycles. Rather, acetylation contributed to seasonal differences, being highest in winter (specifically in torpor), consistent with fasting physiology and decreased abundance of the mitochondrial deacetylase, SIRT3. PMID:24642758

Fasting and refeeding induces changes in the mouse hepatic lipid droplet proteome.

PubMed

Kramer, David A; Quiroga, Ariel D; Lian, Jihong; Fahlman, Richard P; Lehner, Richard

2018-06-15

During fasting, the liver increases lipid storage as a mean to reserve and provide energy for vital cellular functions. After re-feeding, hepatocytes rapidly decrease the amount of triacylglycerol that is stored in lipid droplets (LDs), visible as the size of hepatic LDs significantly decreases after re-feeding. Little is known about the changes in the liver LD proteome that occur during the fasting/re-feeding transition. This study aimed to investigate the hepatic LD proteome in fasted and re-fed conditions in the mouse. Using label-free LC-MS/MS analysis the relative abundance of 817 proteins was determined in highly purified LDs. Comparative analysis for differential protein abundance with respect to feeding states revealed 130 with higher abundance in LDs from fasted mice and 31 in LDs from re-fed mice. Among proteins observed to have higher abundance on LDs in the fasted state we found perilipin-5, and several mitochondrial and peroxisomal marker proteins, supporting the role of LDs in the provision of substrates for fatty acid oxidation. Proteins of higher abundance upon re-feeding included several peroxisomal and mitochondrial marker proteins and expand our understanding of the dynamic nature of the hepatic LD proteome according to the energetic requirements of the cell. Proteomic investigations have been revealing the complexities and dynamics of cellular LDs from a variety of cell types. As these sub-cellular structures are truly dynamic in nature, our investigations reveal that simply the feeding state of an animal leads to significant changes to the protein composition of LDs and suggest a variety of dynamic interactions with other cellular organelles, such as the mitochondria and peroxisomes. As such, the experimental design for investigations of this cellular structure must consider this dynamic baseline. Lastly our analysis on global protein abundance has revealed the unforeseen high abundance of murine major urinary proteins associated with hepatic lipid droplets, which warrants further investigations. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Thermosensitivity of growth is determined by chaperone-mediated proteome reallocation

PubMed Central

Chen, Ke; Gao, Ye; Mih, Nathan; O’Brien, Edward J.; Yang, Laurence; Palsson, Bernhard O.

2017-01-01

Maintenance of a properly folded proteome is critical for bacterial survival at notably different growth temperatures. Understanding the molecular basis of thermoadaptation has progressed in two main directions, the sequence and structural basis of protein thermostability and the mechanistic principles of protein quality control assisted by chaperones. Yet we do not fully understand how structural integrity of the entire proteome is maintained under stress and how it affects cellular fitness. To address this challenge, we reconstruct a genome-scale protein-folding network for Escherichia coli and formulate a computational model, FoldME, that provides statistical descriptions of multiscale cellular response consistent with many datasets. FoldME simulations show (i) that the chaperones act as a system when they respond to unfolding stress rather than achieving efficient folding of any single component of the proteome, (ii) how the proteome is globally balanced between chaperones for folding and the complex machinery synthesizing the proteins in response to perturbation, (iii) how this balancing determines growth rate dependence on temperature and is achieved through nonspecific regulation, and (iv) how thermal instability of the individual protein affects the overall functional state of the proteome. Overall, these results expand our view of cellular regulation, from targeted specific control mechanisms to global regulation through a web of nonspecific competing interactions that modulate the optimal reallocation of cellular resources. The methodology developed in this study enables genome-scale integration of environment-dependent protein properties and a proteome-wide study of cellular stress responses. PMID:29073085

Use of proteomic methods in the analysis of human body fluids in Alzheimer research.

PubMed

Zürbig, Petra; Jahn, Holger

2012-12-01

Proteomics is the study of the entire population of proteins and peptides in an organism or a part of it, such as a cell, tissue, or fluids like cerebrospinal fluid, plasma, serum, urine, or saliva. It is widely assumed that changes in the composition of the proteome may reflect disease states and provide clues to its origin, eventually leading to targets for new treatments. The ability to perform large-scale proteomic studies now is based jointly on recent advances in our analytical methods. Separation techniques like CE and 2DE have developed and matured. Detection methods like MS have also improved greatly in the last 5 years. These developments have also driven the fields of bioinformatics, needed to deal with the increased data production and systems biology. All these developing methods offer specific advantages but also come with certain limitations. This review describes the different proteomic methods used in the field, their limitations, and their possible pitfalls. Based on a literature search in PubMed, we identified 112 studies that applied proteomic techniques to identify biomarkers for Alzheimer disease. This review describes the results of these studies on proteome changes in human body fluids of Alzheimer patients reviewing the most important studies. We extracted a list of 366 proteins and peptides that were identified by these studies as potential targets in Alzheimer research. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

P2P proteomics -- data sharing for enhanced protein identification

PubMed Central

2012-01-01

Background In order to tackle the important and challenging problem in proteomics of identifying known and new protein sequences using high-throughput methods, we propose a data-sharing platform that uses fully distributed P2P technologies to share specifications of peer-interaction protocols and service components. By using such a platform, information to be searched is no longer centralised in a few repositories but gathered from experiments in peer proteomics laboratories, which can subsequently be searched by fellow researchers. Methods The system distributively runs a data-sharing protocol specified in the Lightweight Communication Calculus underlying the system through which researchers interact via message passing. For this, researchers interact with the system through particular components that link to database querying systems based on BLAST and/or OMSSA and GUI-based visualisation environments. We have tested the proposed platform with data drawn from preexisting MS/MS data reservoirs from the 2006 ABRF (Association of Biomolecular Resource Facilities) test sample, which was extensively tested during the ABRF Proteomics Standards Research Group 2006 worldwide survey. In particular we have taken the data available from a subset of proteomics laboratories of Spain's National Institute for Proteomics, ProteoRed, a network for the coordination, integration and development of the Spanish proteomics facilities. Results and Discussion We performed queries against nine databases including seven ProteoRed proteomics laboratories, the NCBI Swiss-Prot database and the local database of the CSIC/UAB Proteomics Laboratory. A detailed analysis of the results indicated the presence of a protein that was supported by other NCBI matches and highly scored matches in several proteomics labs. The analysis clearly indicated that the protein was a relatively high concentrated contaminant that could be present in the ABRF sample. This fact is evident from the information that could be derived from the proposed P2P proteomics system, however it is not straightforward to arrive to the same conclusion by conventional means as it is difficult to discard organic contamination of samples. The actual presence of this contaminant was only stated after the ABRF study of all the identifications reported by the laboratories. PMID:22293032

Core Proteomic Analysis of Unique Metabolic Pathways of Salmonella enterica for the Identification of Potential Drug Targets.

PubMed

Uddin, Reaz; Sufian, Muhammad

2016-01-01

Infections caused by Salmonella enterica, a Gram-negative facultative anaerobic bacteria belonging to the family of Enterobacteriaceae, are major threats to the health of humans and animals. The recent availability of complete genome data of pathogenic strains of the S. enterica gives new avenues for the identification of drug targets and drug candidates. We have used the genomic and metabolic pathway data to identify pathways and proteins essential to the pathogen and absent from the host. We took the whole proteome sequence data of 42 strains of S. enterica and Homo sapiens along with KEGG-annotated metabolic pathway data, clustered proteins sequences using CD-HIT, identified essential genes using DEG database and discarded S. enterica homologs of human proteins in unique metabolic pathways (UMPs) and characterized hypothetical proteins with SVM-prot and InterProScan. Through this core proteomic analysis we have identified enzymes essential to the pathogen. The identification of 73 enzymes common in 42 strains of S. enterica is the real strength of the current study. We proposed all 73 unexplored enzymes as potential drug targets against the infections caused by the S. enterica. The study is comprehensive around S. enterica and simultaneously considered every possible pathogenic strain of S. enterica. This comprehensiveness turned the current study significant since, to the best of our knowledge it is the first subtractive core proteomic analysis of the unique metabolic pathways applied to any pathogen for the identification of drug targets. We applied extensive computational methods to shortlist few potential drug targets considering the druggability criteria e.g. Non-homologous to the human host, essential to the pathogen and playing significant role in essential metabolic pathways of the pathogen (i.e. S. enterica). In the current study, the subtractive proteomics through a novel approach was applied i.e. by considering only proteins of the unique metabolic pathways of the pathogens and mining the proteomic data of all completely sequenced strains of the pathogen, thus improving the quality and application of the results. We believe that the sharing of the knowledge from this study would eventually lead to bring about novel and unique therapeutic regimens against the infections caused by the S. enterica.

Final Report: Proteomic study of brassinosteroid responses in Arabidopsis

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

Wang, Zhiyong; Burlingame, Alma

2017-11-29

The steroid hormone brassinosteroid (BR) is a major growth-promoting phytohormone. The specific aim of the current project is to identify BR-regulated proteins and characterize their functions in various aspects of plant growth, development, and adaptation. Our research has significantly advanced our understanding of how BR signal is transduced from the receptor at the cell surface to changes of nuclear gene expression and other cellular responses such as vesicle trafficking, as well as developmental transitions such as seed germination and flowering. We have also developed effective proteomic methods for quantitative analysis of protein phosphorylation and for identification of glycosylated proteins. Throughmore » this DOE funding, we have performed several proteomic experiments and made major discoveries.« less

Proteomic technology for biomarker profiling in cancer: an update*

PubMed Central

Alaoui-Jamali, Moulay A.; Xu, Ying-jie

2006-01-01

The progress in the understanding of cancer progression and early detection has been slow and frustrating due to the complex multifactorial nature and heterogeneity of the cancer syndrome. To date, no effective treatment is available for advanced cancers, which remain a major cause of morbidity and mortality. Clearly, there is urgent need to unravel novel biomarkers for early detection. Most of the functional information of the cancer-associated genes resides in the proteome. The later is an exceptionally complex biological system involving several proteins that function through posttranslational modifications and dynamic intermolecular collisions with partners. These protein complexes can be regulated by signals emanating from cancer cells, their surrounding tissue microenvironment, and/or from the host. Some proteins are secreted and/or cleaved into the extracellular milieu and may represent valuable serum biomarkers for diagnosis purpose. It is estimated that the cancer proteome may include over 1.5 million proteins as a result of posttranslational processing and modifications. Such complexity clearly highlights the need for ultra-high resolution proteomic technology for robust quantitative protein measurements and data acquisition. This review is to update the current research efforts in high-resolution proteomic technology for discovery and monitoring cancer biomarkers. PMID:16625706

Accounting for isotopic clustering in Fourier transform mass spectrometry data analysis for clinical diagnostic studies.

PubMed

Kakourou, Alexia; Vach, Werner; Nicolardi, Simone; van der Burgt, Yuri; Mertens, Bart

2016-10-01

Mass spectrometry based clinical proteomics has emerged as a powerful tool for high-throughput protein profiling and biomarker discovery. Recent improvements in mass spectrometry technology have boosted the potential of proteomic studies in biomedical research. However, the complexity of the proteomic expression introduces new statistical challenges in summarizing and analyzing the acquired data. Statistical methods for optimally processing proteomic data are currently a growing field of research. In this paper we present simple, yet appropriate methods to preprocess, summarize and analyze high-throughput MALDI-FTICR mass spectrometry data, collected in a case-control fashion, while dealing with the statistical challenges that accompany such data. The known statistical properties of the isotopic distribution of the peptide molecules are used to preprocess the spectra and translate the proteomic expression into a condensed data set. Information on either the intensity level or the shape of the identified isotopic clusters is used to derive summary measures on which diagnostic rules for disease status allocation will be based. Results indicate that both the shape of the identified isotopic clusters and the overall intensity level carry information on the class outcome and can be used to predict the presence or absence of the disease.

Advances in Quantitative Proteomics of Microbes and Microbial Communities

NASA Astrophysics Data System (ADS)

Waldbauer, J.; Zhang, L.; Rizzo, A. I.

2015-12-01

Quantitative measurements of gene expression are key to developing a mechanistic, predictive understanding of how microbial metabolism drives many biogeochemical fluxes and responds to environmental change. High-throughput RNA-sequencing can afford a wealth of information about transcript-level expression patterns, but it is becoming clear that expression dynamics are often very different at the protein level where biochemistry actually occurs. These divergent dynamics between levels of biological organization necessitate quantitative proteomic measurements to address many biogeochemical questions. The protein-level expression changes that underlie shifts in the magnitude, or even the direction, of metabolic and biogeochemical fluxes can be quite subtle and test the limits of current quantitative proteomics techniques. Here we describe methodologies for high-precision, whole-proteome quantification that are applicable to both model organisms of biogeochemical interest that may not be genetically tractable, and to complex community samples from natural environments. Employing chemical derivatization of peptides with multiple isotopically-coded tags, this strategy is rapid and inexpensive, can be implemented on a wide range of mass spectrometric instrumentation, and is relatively insensitive to chromatographic variability. We demonstrate the utility of this quantitative proteomics approach in application to both isolates and natural communities of sulfur-metabolizing and photosynthetic microbes.

Quantitative proteomics in Giardia duodenalis-Achievements and challenges.

PubMed

Emery, Samantha J; Lacey, Ernest; Haynes, Paul A

2016-08-01

Giardia duodenalis (syn. G. lamblia and G. intestinalis) is a protozoan parasite of vertebrates and a major contributor to the global burden of diarrheal diseases and gastroenteritis. The publication of multiple genome sequences in the G. duodenalis species complex has provided important insights into parasite biology, and made post-genomic technologies, including proteomics, significantly more accessible. The aims of proteomics are to identify and quantify proteins present in a cell, and assign functions to them within the context of dynamic biological systems. In Giardia, proteomics in the post-genomic era has transitioned from reliance on gel-based systems to utilisation of a diverse array of techniques based on bottom-up LC-MS/MS technologies. Together, these have generated crucial foundations for subcellular proteomes, elucidated intra- and inter-assemblage isolate variation, and identified pathways and markers in differentiation, host-parasite interactions and drug resistance. However, in Giardia, proteomics remains an emerging field, with considerable shortcomings evident from the published research. These include a bias towards assemblage A, a lack of emphasis on quantitative analytical techniques, and limited information on post-translational protein modifications. Additionally, there are multiple areas of research for which proteomic data is not available to add value to published transcriptomic data. The challenge of amalgamating data in the systems biology paradigm necessitates the further generation of large, high-quality quantitative datasets to accurately model parasite biology. This review surveys the current proteomic research available for Giardia and evaluates their technical and quantitative approaches, while contextualising their biological insights into parasite pathology, isolate variation and eukaryotic evolution. Finally, we propose areas of priority for the generation of future proteomic data to explore fundamental questions in Giardia, including the analysis of post-translational modifications, and the design of MS-based assays for validation of differentially expressed proteins in large datasets. Copyright © 2016 Elsevier B.V. All rights reserved.

neXtProt: organizing protein knowledge in the context of human proteome projects.

PubMed

Gaudet, Pascale; Argoud-Puy, Ghislaine; Cusin, Isabelle; Duek, Paula; Evalet, Olivier; Gateau, Alain; Gleizes, Anne; Pereira, Mario; Zahn-Zabal, Monique; Zwahlen, Catherine; Bairoch, Amos; Lane, Lydie

2013-01-04

About 5000 (25%) of the ~20400 human protein-coding genes currently lack any experimental evidence at the protein level. For many others, there is only little information relative to their abundance, distribution, subcellular localization, interactions, or cellular functions. The aim of the HUPO Human Proteome Project (HPP, www.thehpp.org ) is to collect this information for every human protein. HPP is based on three major pillars: mass spectrometry (MS), antibody/affinity capture reagents (Ab), and bioinformatics-driven knowledge base (KB). To meet this objective, the Chromosome-Centric Human Proteome Project (C-HPP) proposes to build this catalog chromosome-by-chromosome ( www.c-hpp.org ) by focusing primarily on proteins that currently lack MS evidence or Ab detection. These are termed "missing proteins" by the HPP consortium. The lack of observation of a protein can be due to various factors including incorrect and incomplete gene annotation, low or restricted expression, or instability. neXtProt ( www.nextprot.org ) is a new web-based knowledge platform specific for human proteins that aims to complement UniProtKB/Swiss-Prot ( www.uniprot.org ) with detailed information obtained from carefully selected high-throughput experiments on genomic variation, post-translational modifications, as well as protein expression in tissues and cells. This article describes how neXtProt contributes to prioritize C-HPP efforts and integrates C-HPP results with other research efforts to create a complete human proteome catalog.

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 .

2016 update of the PRIDE database and its related tools

PubMed Central

Vizcaíno, Juan Antonio; Csordas, Attila; del-Toro, Noemi; Dianes, José A.; Griss, Johannes; Lavidas, Ilias; Mayer, Gerhard; Perez-Riverol, Yasset; Reisinger, Florian; Ternent, Tobias; Xu, Qing-Wei; Wang, Rui; Hermjakob, Henning

2016-01-01

The PRoteomics IDEntifications (PRIDE) database is one of the world-leading data repositories of mass spectrometry (MS)-based proteomics data. Since the beginning of 2014, PRIDE Archive (http://www.ebi.ac.uk/pride/archive/) is the new PRIDE archival system, replacing the original PRIDE database. Here we summarize the developments in PRIDE resources and related tools since the previous update manuscript in the Database Issue in 2013. PRIDE Archive constitutes a complete redevelopment of the original PRIDE, comprising a new storage backend, data submission system and web interface, among other components. PRIDE Archive supports the most-widely used PSI (Proteomics Standards Initiative) data standard formats (mzML and mzIdentML) and implements the data requirements and guidelines of the ProteomeXchange Consortium. The wide adoption of ProteomeXchange within the community has triggered an unprecedented increase in the number of submitted data sets (around 150 data sets per month). We outline some statistics on the current PRIDE Archive data contents. We also report on the status of the PRIDE related stand-alone tools: PRIDE Inspector, PRIDE Converter 2 and the ProteomeXchange submission tool. Finally, we will give a brief update on the resources under development ‘PRIDE Cluster’ and ‘PRIDE Proteomes’, which provide a complementary view and quality-scored information of the peptide and protein identification data available in PRIDE Archive. PMID:26527722

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

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.

Proteomics: A valuable approach to elucidate spermatozoa post -testicular maturation in the endangered Acipenseridae family.

PubMed

Boccaletto, Pietro; Siddique, Mohammad Abdul Momin; Cosson, Jacky

2018-05-01

Proteomics techniques, such as two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, and differential gel electrophoresis, have been extensively used to describe the protein composition of male gametes in different animals, mainly mammals. They have also provided a deeper understanding of protein functions involved in sperm processes, as in processes that in humans lead to male infertility. However, few studies focus on fish sperm proteomics and even fewer have tried to explore the proteomic profile of Sturgeon spermatozoa. Sturgeon is an endangered, ancient group of fish species exploited mostly for caviar. In this fish group, a part of the process that leads to final functional maturation of spermatozoa so as to have the capability to activate eggs during the fertilization process. This process has a broad similarity to post-testicular maturation in mammals; where spermatozoa leaving the testes must be mixed with seminal fluid along the transit through the Wolffian ducts to modify its surface membrane protein composition, leading to axonemal and acrosomal competence. The aim of this study was to review the current literature on various proteomic techniques, their usefulness in separating, identifying and studying the proteome composition of the fish spermatozoon, as well as their potential applications in studying the post-testicular maturation process in Sturgeon. Such understanding could lead to development of more sophisticated aquaculture techniques, favorable for sturgeon reproduction. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Myeloperoxidase mediated HDL oxidation and HDL proteome changes do not contribute to dysfunctional HDL in Chinese subjects with coronary artery disease

PubMed Central

Yu, Haiyi; Li, Lei; He, Liyun; Gao, Wei; Liu, Xiaodan; Guo, Yanhong; Byun, Jaeman; Zhang, Jifeng; Chen, Y. Eugene

2018-01-01

High density lipoprotein (HDL) cholesterol levels and cholesterol efflux capacity (CEC) are inversely correlated with coronary artery disease (CAD) risk. Myeloperoxidase (MPO) derived oxidants and HDL proteome changes are implicated in HDL dysfunction in subjects with CAD in the United States; however, the effect of MPO on HDL function and HDL proteome in ethnic Chinese population is unknown. We recruited four matched ethnic Chinese groups (20 patients each): subjects with 1) low HDL levels (HDL levels in men <40mg/dL and women <50mg/dL) and non-CAD (identified by coronary angiography or cardiac CT angiography); 2) low HDL and CAD; 3) high HDL (men >50mg/dL; women >60mg/dL) with no CAD; and 4) high HDL with CAD. Serum cytokines, serum MPO levels, serum CEC, MPO-oxidized HDL tyrosine moieties, and HDL proteome were assessed by mass spectrometry individually in the four groups. The cytokines, MPO levels, and HDL proteome profiles were not significantly different between the four groups. As expected, CEC was depressed in the entire CAD group but more specifically in the CAD low-HDL group. HDL of CAD subjects had significantly higher 3-nitrotyrosine than non-CAD subjects, but the MPO-specific 3-chlorotyrosine was unchanged; CEC in the CAD low-HDL group did not correlate with either HDL 3-chlorotyrosine or 3-nitrotyrosine levels. Neither 3-chlorotyrosine, which is MPO-specific, nor 3-nitrotyrosine generated from MPO or other reactive nitrogen species was associated with CEC. MPO mediated oxidative stress and HDL proteome composition changes are not the primary cause HDL dysfunction in Chinese subjects with CAD. These studies highlight ethnic differences in HDL dysfunction between United States and Chinese cohorts raising possibility of unique pathways of HDL dysfunction in this cohort. PMID:29505607

Tear film proteome in age-related macular degeneration.

PubMed

Winiarczyk, Mateusz; Kaarniranta, Kai; Winiarczyk, Stanisław; Adaszek, Łukasz; Winiarczyk, Dagmara; Mackiewicz, Jerzy

2018-06-01

Age-related macular degeneration (AMD) is the main reason for blindness in elderly people in the developed countries. Current screening protocols have limitations in detecting the early signs of retinal degeneration. Therefore, it would be desirable to find novel biomarkers for early detection of AMD. Development of novel biomarkers would help in the prevention, diagnostics, and treatment of AMD. Proteomic analysis of tear film has shown promise in this research area. If an optimal set of biomarkers could be obtained from accessible body fluids, it would represent a reliable way to monitor disease progression and response to novel therapies. Tear films were collected on Schirmer strips from a total of 22 patients (8 with wet AMD, 6 with dry AMD, and 8 control individuals). 2D electrophoresis was used to separate tear film proteins prior to their identification with matrix-assisted laser desorption/ionization time of flight spectrometer (MALDI-TOF/TOF) and matching with functional databases. A total of 342 proteins were identified. Most of them were previously described in various proteomic studies concerning AMD. Shootin-1, histatin-3, fidgetin-like protein 1, SRC kinase signaling inhibitor, Graves disease carrier protein, actin cytoplasmic 1, prolactin-inducible protein 1, and protein S100-A7A were upregulated in the tear film samples isolated from AMD patients and were not previously linked with this disease in any proteomic analysis. The upregulated proteins supplement our current knowledge of AMD pathogenesis, providing evidence that certain specific proteins are expressed into the tear film in AMD. As far we are aware, this is the first study to have undertaken a comprehensive in-depth analysis of the human tear film proteome in AMD patients.

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

Elzek, Mohamed A.; Rodland, Karin D.

In the past decade, there has been an increasing interest in applying proteomics to assist in understanding the pathogenesis of ovarian cancer, elucidating the mechanism of drug resistance, and in the development of biomarkers for early detection of ovarian cancer. Although ovarian cancer is a spectrum of different diseases, the strategies for diagnosis and treatment with surgery and adjuvant therapy are similar across ovarian cancer types, increasing the general applicability of discoveries made through proteomics research. While proteomic experiments face many difficulties which slow the pace of clinical applications, recent advances in proteomic technology contribute significantly to the identification ofmore » aberrant proteins and networks which can serve as targets for biomarker development and individualized therapies. This review provides a summary of the literature on proteomics’ contributions to ovarian cancer research and highlights the current issues, future directions, and challenges. In conclusion, we propose that protein-level characterization of primary lesion in ovarian cancer can decipher the mystery of this disease, improve diagnostic tools, and lead to more effective screening programs.« less

P-MartCancer–Interactive Online Software to Enable Analysis of Shotgun Cancer Proteomic Datasets

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

Webb-Robertson, Bobbie-Jo M.; Bramer, Lisa M.; Jensen, Jeffrey L.

P-MartCancer is a new interactive web-based software environment that enables biomedical and biological scientists to perform in-depth analyses of global proteomics data without requiring direct interaction with the data or with statistical software. P-MartCancer offers a series of statistical modules associated with quality assessment, peptide and protein statistics, protein quantification and exploratory data analyses driven by the user via customized workflows and interactive visualization. Currently, P-MartCancer offers access to multiple cancer proteomic datasets generated through the Clinical Proteomics Tumor Analysis Consortium (CPTAC) at the peptide, gene and protein levels. P-MartCancer is deployed using Azure technologies (http://pmart.labworks.org/cptac.html), the web-service is alternativelymore » available via Docker Hub (https://hub.docker.com/r/pnnl/pmart-web/) and many statistical functions can be utilized directly from an R package available on GitHub (https://github.com/pmartR).« less

Systems biology, proteomics, and the future of health care: toward predictive, preventative, and personalized medicine.

PubMed

Weston, Andrea D; Hood, Leroy

2004-01-01

The emergence of systems biology is bringing forth a new set of challenges for advancing science and technology. Defining ways of studying biological systems on a global level, integrating large and disparate data types, and dealing with the infrastructural changes necessary to carry out systems biology, are just a few of the extraordinary tasks of this growing discipline. Despite these challenges, the impact of systems biology will be far-reaching, and significant progress has already been made. Moving forward, the issue of how to use systems biology to improve the health of individuals must be a priority. It is becoming increasingly apparent that the field of systems biology and one of its important disciplines, proteomics, will have a major role in creating a predictive, preventative, and personalized approach to medicine. In this review, we define systems biology, discuss the current capabilities of proteomics and highlight some of the necessary milestones for moving systems biology and proteomics into mainstream health care.

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.

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

Characterization of the Low-Molecular-Weight Human Plasma Peptidome.

PubMed

Greening, David W; Simpson, Richard J

2017-01-01

The human plasma proteome represents an important secreted sub-proteome. Proteomic analysis of blood plasma with mass spectrometry is a challenging task. The high complexity and wide dynamic range of proteins as well as the presence of several proteins at very high concentrations complicate the profiling of the human plasma proteome. The peptidome (or low-molecular-weight fraction, LMF) of the human plasma proteome is an invaluable source of biological information, especially in the context of identifying plasma-based markers of disease. Peptides are generated by active synthesis and proteolytic processing, often yielding proteolytic fragments that mediate a variety of physiological and pathological functions. As such, degradomic studies, investigating cleavage products via peptidomics and top-down proteomics in particular, have warranted significant research interest. However, due to their molecular weight, abundance, and solubility, issues with identifying specific cleavage sites and coverage of peptide fragments remain challenging. Peptidomics is currently focused toward comprehensively studying peptides cleaved from precursor proteins by endogenous proteases. This protocol outlines a standardized rapid and reproducible procedure for peptidomic profiling of human plasma using centrifugal ultrafiltration and mass spectrometry. Ultrafiltration is a convective process that uses anisotropic semipermeable membranes to separate macromolecular species on the basis of size. We have optimized centrifugal ultrafiltration (cellulose triacetate membrane) for plasma fractionation with respect to buffer and solvent composition, centrifugal force, duration, and temperature to facilitate recovery >95% and enrichment of the human plasma peptidome. This method serves as a comprehensive and facile process to enrich and identify a key, underrepresented sub-proteome of human blood plasma.

The Human Skeletal Muscle Proteome Project: a reappraisal of the current literature

PubMed Central

Gonzalez‐Freire, Marta; Semba, Richard D.; Ubaida‐Mohien, Ceereena; Fabbri, Elisa; Scalzo, Paul; Højlund, Kurt; Dufresne, Craig; Lyashkov, Alexey

2016-01-01

Abstract Skeletal muscle is a large organ that accounts for up to half the total mass of the human body. A progressive decline in muscle mass and strength occurs with ageing and in some individuals configures the syndrome of ‘sarcopenia’, a condition that impairs mobility, challenges autonomy, and is a risk factor for mortality. The mechanisms leading to sarcopenia as well as myopathies are still little understood. The Human Skeletal Muscle Proteome Project was initiated with the aim to characterize muscle proteins and how they change with ageing and disease. We conducted an extensive review of the literature and analysed publically available protein databases. A systematic search of peer‐reviewed studies was performed using PubMed. Search terms included ‘human’, ‘skeletal muscle’, ‘proteome’, ‘proteomic(s)’, and ‘mass spectrometry’, ‘liquid chromatography‐mass spectrometry (LC‐MS/MS)’. A catalogue of 5431 non‐redundant muscle proteins identified by mass spectrometry‐based proteomics from 38 peer‐reviewed scientific publications from 2002 to November 2015 was created. We also developed a nosology system for the classification of muscle proteins based on localization and function. Such inventory of proteins should serve as a useful background reference for future research on changes in muscle proteome assessed by quantitative mass spectrometry‐based proteomic approaches that occur with ageing and diseases. This classification and compilation of the human skeletal muscle proteome can be used for the identification and quantification of proteins in skeletal muscle to discover new mechanisms for sarcopenia and specific muscle diseases that can be targeted for the prevention and treatment. PMID:27897395

Boosting the globalization of plant proteomics through INPPO: current developments and future prospects.

PubMed

Agrawal, Ganesh Kumar; Sarkar, Abhijit; Agrawal, Raj; Ndimba, Bongani Kaiser; Tanou, Georgia; Dunn, Michael J; Kieselbach, Thomas; Cramer, Rainer; Wienkoop, Stefanie; Chen, Sixue; Rafudeen, Mohammed Suhail; Deswal, Renu; Barkla, Bronwyn J; Weckwerth, Wolfram; Heazlewood, Joshua L; Renaut, Jenny; Job, Dominique; Chakraborty, Niranjan; Rakwal, Randeep

2012-02-01

The International Plant Proteomics Organization (INPPO) is a non-profit-organization consisting of people who are involved or interested in plant proteomics. INPPO is constantly growing in volume and activity, which is mostly due to the realization among plant proteomics researchers worldwide for the need of such a global platform. Their active participation resulted in the rapid growth within the first year of INPPO's official launch in 2011 via its website (www.inppo.com) and publication of the 'Viewpoint paper' in a special issue of PROTEOMICS (May 2011). Here, we will be highlighting the progress achieved in the year 2011 and the future targets for the year 2012 and onwards. INPPO has achieved a successful administrative structure, the Core Committee (CC; composed of President, Vice-President, and General Secretaries), Executive Council (EC), and General Body (GB) to achieve INPPO objectives. Various committees and subcommittees are in the process of being functionalized via discussion amongst scientists around the globe. INPPO's primary aim to popularize the plant proteomics research in biological sciences has also been recognized by PROTEOMICS where a section dedicated to plant proteomics has been introduced starting January 2012, following the very first issue of this journal devoted to plant proteomics in May 2011. To disseminate organizational activities to the scientific community, INPPO has launched a biannual (in January and July) newsletter entitled 'INPPO Express: News & Views' with the first issue published in January 2012. INPPO is also planning to have several activities in 2012, including programs within the Education Outreach committee in different countries, and the development of research ideas and proposals with priority on crop and horticultural plants, while keeping tight interactions with proteomics programs on model plants such as Arabidopsis thaliana, rice, and Medicago truncatula. Altogether, the INPPO progress and upcoming activities are because of immense support, dedication, and hard work of all members of the INPPO community, and also due to the wide encouragement and support from the communities (scientific and non-scientific). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

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.

Open source libraries and frameworks for mass spectrometry based proteomics: A developer's perspective☆

PubMed Central

Perez-Riverol, Yasset; Wang, Rui; Hermjakob, Henning; Müller, Markus; Vesada, Vladimir; Vizcaíno, Juan Antonio

2014-01-01

Data processing, management and visualization are central and critical components of a state of the art high-throughput mass spectrometry (MS)-based proteomics experiment, and are often some of the most time-consuming steps, especially for labs without much bioinformatics support. The growing interest in the field of proteomics has triggered an increase in the development of new software libraries, including freely available and open-source software. From database search analysis to post-processing of the identification results, even though the objectives of these libraries and packages can vary significantly, they usually share a number of features. Common use cases include the handling of protein and peptide sequences, the parsing of results from various proteomics search engines output files, and the visualization of MS-related information (including mass spectra and chromatograms). In this review, we provide an overview of the existing software libraries, open-source frameworks and also, we give information on some of the freely available applications which make use of them. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan. PMID:23467006

Open source libraries and frameworks for mass spectrometry based proteomics: a developer's perspective.

PubMed

Perez-Riverol, Yasset; Wang, Rui; Hermjakob, Henning; Müller, Markus; Vesada, Vladimir; Vizcaíno, Juan Antonio

2014-01-01

Data processing, management and visualization are central and critical components of a state of the art high-throughput mass spectrometry (MS)-based proteomics experiment, and are often some of the most time-consuming steps, especially for labs without much bioinformatics support. The growing interest in the field of proteomics has triggered an increase in the development of new software libraries, including freely available and open-source software. From database search analysis to post-processing of the identification results, even though the objectives of these libraries and packages can vary significantly, they usually share a number of features. Common use cases include the handling of protein and peptide sequences, the parsing of results from various proteomics search engines output files, and the visualization of MS-related information (including mass spectra and chromatograms). In this review, we provide an overview of the existing software libraries, open-source frameworks and also, we give information on some of the freely available applications which make use of them. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan. Copyright © 2013 Elsevier B.V. All rights reserved.

Potential for proteomic approaches in determining efficacy biomarkers following administration of fish oils rich in omega-3 fatty acids: application in pancreatic cancers.

PubMed

Runau, Franscois; Arshad, Ali; Isherwood, John; Norris, Leonie; Howells, Lynne; Metcalfe, Matthew; Dennison, Ashley

2015-06-01

Pancreatic cancer is a disease with a significantly poor prognosis. Despite modern advances in other medical, surgical, and oncologic therapy, the outcome from pancreatic cancer has improved little over the last 40 years. To improve the management of this difficult disease, trials investigating the use of dietary and parenteral fish oils rich in omega-3 (ω-3) fatty acids, exhibiting proven anti-inflammatory and anticarcinogenic properties, have revealed favorable results in pancreatic cancers. Proteomics is the large-scale study of proteins that attempts to characterize the complete set of proteins encoded by the genome of an organism and that, with the use of sensitive mass spectrometric-based techniques, has allowed high-throughput analysis of the proteome to aid identification of putative biomarkers pertinent to given disease states. These biomarkers provide useful insight into potentially discovering new markers for early detection or elucidating the efficacy of treatment on pancreatic cancers. Here, our review identifies potential proteomic-based biomarkers in pancreatic cancer relating to apoptosis, cell proliferation, angiogenesis, and metabolic regulation in clinical studies. We also reviewed proteomic biomarkers from the administration of ω-3 fatty acids that act on similar anticarcinogenic pathways as above and reflect that proteomic studies on the effect of ω-3 fatty acids in pancreatic cancer will yield favorable results. © 2015 American Society for Parenteral and Enteral Nutrition.

Development of a proteomic approach to monitor protein synthesis in mycotoxin producing moulds.

PubMed

Milles, J; Krämer, J; Prange, A

2007-12-01

In general, proteome studies compare different states of metabolism to investigate external or internal influences on protein expression. In the context of mycotoxin production the method could open another view on this complex and could be helpful to gain knowledge about proteins which are involved in metabolism (enzymes, transporters). In this short technical report, we describe a new protocol suitable for protein preparation for whole proteome analysis ofFusarium graminearum. Cell lysis was performed by grinding the mycelium with liquid nitrogen. Proteins were extracted with TCA/acetone and then cleaned; the isolated proteins were separated in a 2D-gel electrophoresis system (BioRad) using different pH gradients. The protocol established seems also generally applicable for other mycotoxin producing fungi.

Review of Software Tools for Design and Analysis of Large scale MRM Proteomic Datasets

PubMed Central

Colangelo, Christopher M.; Chung, Lisa; Bruce, Can; Cheung, Kei-Hoi

2013-01-01

Selective or Multiple Reaction monitoring (SRM/MRM) is a liquid-chromatography (LC)/tandem-mass spectrometry (MS/MS) method that enables the quantitation of specific proteins in a sample by analyzing precursor ions and the fragment ions of their selected tryptic peptides. Instrumentation software has advanced to the point that thousands of transitions (pairs of primary and secondary m/z values) can be measured in a triple quadrupole instrument coupled to an LC, by a well-designed scheduling and selection of m/z windows. The design of a good MRM assay relies on the availability of peptide spectra from previous discovery-phase LC-MS/MS studies. The tedious aspect of manually developing and processing MRM assays involving thousands of transitions has spurred to development of software tools to automate this process. Software packages have been developed for project management, assay development, assay validation, data export, peak integration, quality assessment, and biostatistical analysis. No single tool provides a complete end-to-end solution, thus this article reviews the current state and discusses future directions of these software tools in order to enable researchers to combine these tools for a comprehensive targeted proteomics workflow. PMID:23702368

Unraveling the molecular repertoire of tears as a source of biomarkers: beyond ocular diseases.

PubMed

Pieragostino, Damiana; D'Alessandro, Michele; di Ioia, Maria; Di Ilio, Carmine; Sacchetta, Paolo; Del Boccio, Piero

2015-02-01

Proteomics and metabolomics investigations of body fluids present several challenges for biomarker discovery of several diseases. The search for biomarkers is actually conducted in different body fluids, even if the ideal biomarker can be found in an easily accessible biological fluid, because, if validated, the biomarker could be sought in the healthy population. In this regard, tears could be considered an optimum material obtained by noninvasive procedures. In the past years, the scientific community has become more interested in the study of tears for the research of new biomarkers not only for ocular diseases. In this review, we provide a discussion on the current state of biomarkers research in tears and their relevance for clinical practice, and report the main results of clinical proteomics studies on systemic and eye diseases. We summarize the main methods for tear samples analyses and report recent advances in "omics" platforms for tears investigations. Moreover, we want to take stock of the emerging field of metabolomics and lipidomics as a new and integrated approach to study protein-metabolites interplay for biomarkers research, where tears represent a still unexplored and attractive field. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein and gene model inference based on statistical modeling in k-partite graphs.

PubMed

Gerster, Sarah; Qeli, Ermir; Ahrens, Christian H; Bühlmann, Peter

2010-07-06

One of the major goals of proteomics is the comprehensive and accurate description of a proteome. Shotgun proteomics, the method of choice for the analysis of complex protein mixtures, requires that experimentally observed peptides are mapped back to the proteins they were derived from. This process is also known as protein inference. We present Markovian Inference of Proteins and Gene Models (MIPGEM), a statistical model based on clearly stated assumptions to address the problem of protein and gene model inference for shotgun proteomics data. In particular, we are dealing with dependencies among peptides and proteins using a Markovian assumption on k-partite graphs. We are also addressing the problems of shared peptides and ambiguous proteins by scoring the encoding gene models. Empirical results on two control datasets with synthetic mixtures of proteins and on complex protein samples of Saccharomyces cerevisiae, Drosophila melanogaster, and Arabidopsis thaliana suggest that the results with MIPGEM are competitive with existing tools for protein inference.

A draft map of the mouse pluripotent stem cell spatial proteome

PubMed Central

Christoforou, Andy; Mulvey, Claire M.; Breckels, Lisa M.; Geladaki, Aikaterini; Hurrell, Tracey; Hayward, Penelope C.; Naake, Thomas; Gatto, Laurent; Viner, Rosa; Arias, Alfonso Martinez; Lilley, Kathryn S.

2016-01-01

Knowledge of the subcellular distribution of proteins is vital for understanding cellular mechanisms. Capturing the subcellular proteome in a single experiment has proven challenging, with studies focusing on specific compartments or assigning proteins to subcellular niches with low resolution and/or accuracy. Here we introduce hyperLOPIT, a method that couples extensive fractionation, quantitative high-resolution accurate mass spectrometry with multivariate data analysis. We apply hyperLOPIT to a pluripotent stem cell population whose subcellular proteome has not been extensively studied. We provide localization data on over 5,000 proteins with unprecedented spatial resolution to reveal the organization of organelles, sub-organellar compartments, protein complexes, functional networks and steady-state dynamics of proteins and unexpected subcellular locations. The method paves the way for characterizing the impact of post-transcriptional and post-translational modification on protein location and studies involving proteome-level locational changes on cellular perturbation. An interactive open-source resource is presented that enables exploration of these data. PMID:26754106

Applications of mass spectrometry for quantitative protein analysis in formalin-fixed paraffin-embedded tissues

PubMed Central

Steiner, Carine; Ducret, Axel; Tille, Jean-Christophe; Thomas, Marlene; McKee, Thomas A; Rubbia-Brandt, Laura A; Scherl, Alexander; Lescuyer, Pierre; Cutler, Paul

2014-01-01

Proteomic analysis of tissues has advanced in recent years as instruments and methodologies have evolved. The ability to retrieve peptides from formalin-fixed paraffin-embedded tissues followed by shotgun or targeted proteomic analysis is offering new opportunities in biomedical research. In particular, access to large collections of clinically annotated samples should enable the detailed analysis of pathologically relevant tissues in a manner previously considered unfeasible. In this paper, we review the current status of proteomic analysis of formalin-fixed paraffin-embedded tissues with a particular focus on targeted approaches and the potential for this technique to be used in clinical research and clinical diagnosis. We also discuss the limitations and perspectives of the technique, particularly with regard to application in clinical diagnosis and drug discovery. PMID:24339433

Brucella proteomes--a review.

PubMed

DelVecchio, Vito G; Wagner, Mary Ann; Eschenbrenner, Michel; Horn, Troy A; Kraycer, Jo Ann; Estock, Frank; Elzer, Phil; Mujer, Cesar V

2002-12-20

The proteomes of selected Brucella spp. have been extensively analyzed by utilizing current proteomic technology involving 2-DE and MALDI-MS. In Brucella melitensis, more than 500 proteins were identified. The rapid and large-scale identification of proteins in this organism was accomplished by using the annotated B. melitensis genome which is now available in the GenBank. Coupled with new and powerful tools for data analysis, differentially expressed proteins were identified and categorized into several classes. A global overview of protein expression patterns emerged, thereby facilitating the simultaneous analysis of different metabolic pathways in B. melitensis. Such a global characterization would not have been possible by using time consuming and traditional biochemical approaches. The era of post-genomic technology offers new and exciting opportunities to understand the complete biology of different Brucella species.

Solid-Phase Extraction Strategies to Surmount Body Fluid Sample Complexity in High-Throughput Mass Spectrometry-Based Proteomics

PubMed Central

Bladergroen, Marco R.; van der Burgt, Yuri E. M.

2015-01-01

For large-scale and standardized applications in mass spectrometry- (MS-) based proteomics automation of each step is essential. Here we present high-throughput sample preparation solutions for balancing the speed of current MS-acquisitions and the time needed for analytical workup of body fluids. The discussed workflows reduce body fluid sample complexity and apply for both bottom-up proteomics experiments and top-down protein characterization approaches. Various sample preparation methods that involve solid-phase extraction (SPE) including affinity enrichment strategies have been automated. Obtained peptide and protein fractions can be mass analyzed by direct infusion into an electrospray ionization (ESI) source or by means of matrix-assisted laser desorption ionization (MALDI) without further need of time-consuming liquid chromatography (LC) separations. PMID:25692071

Derivative component analysis for mass spectral serum proteomic profiles.

PubMed

Han, Henry

2014-01-01

As a promising way to transform medicine, mass spectrometry based proteomics technologies have seen a great progress in identifying disease biomarkers for clinical diagnosis and prognosis. However, there is a lack of effective feature selection methods that are able to capture essential data behaviors to achieve clinical level disease diagnosis. Moreover, it faces a challenge from data reproducibility, which means that no two independent studies have been found to produce same proteomic patterns. Such reproducibility issue causes the identified biomarker patterns to lose repeatability and prevents it from real clinical usage. In this work, we propose a novel machine-learning algorithm: derivative component analysis (DCA) for high-dimensional mass spectral proteomic profiles. As an implicit feature selection algorithm, derivative component analysis examines input proteomics data in a multi-resolution approach by seeking its derivatives to capture latent data characteristics and conduct de-noising. We further demonstrate DCA's advantages in disease diagnosis by viewing input proteomics data as a profile biomarker via integrating it with support vector machines to tackle the reproducibility issue, besides comparing it with state-of-the-art peers. Our results show that high-dimensional proteomics data are actually linearly separable under proposed derivative component analysis (DCA). As a novel multi-resolution feature selection algorithm, DCA not only overcomes the weakness of the traditional methods in subtle data behavior discovery, but also suggests an effective resolution to overcoming proteomics data's reproducibility problem and provides new techniques and insights in translational bioinformatics and machine learning. The DCA-based profile biomarker diagnosis makes clinical level diagnostic performances reproducible across different proteomic data, which is more robust and systematic than the existing biomarker discovery based diagnosis. Our findings demonstrate the feasibility and power of the proposed DCA-based profile biomarker diagnosis in achieving high sensitivity and conquering the data reproducibility issue in serum proteomics. Furthermore, our proposed derivative component analysis suggests the subtle data characteristics gleaning and de-noising are essential in separating true signals from red herrings for high-dimensional proteomic profiles, which can be more important than the conventional feature selection or dimension reduction. In particular, our profile biomarker diagnosis can be generalized to other omics data for derivative component analysis (DCA)'s nature of generic data analysis.

Evaluation of the salivary proteome as a surrogate tissue for systems biology approaches to understanding appetite.

PubMed

Harden, Charlotte J; Perez-Carrion, Kristine; Babakordi, Zara; Plummer, Sue F; Hepburn, Natalie; Barker, Margo E; Wright, Phillip C; Evans, Caroline A; Corfe, Bernard M

2012-06-06

Current measurement of appetite depends upon tools that are either subjective (visual analogue scales), or invasive (blood). Saliva is increasingly recognised as a valuable resource for biomarker analysis. Proteomics workflows may provide alternative means for the assessment of appetitive response. The study aimed to assess the potential value of the salivary proteome to detect novel biomarkers of appetite using an iTRAQ-based workflow. Diurnal variation of salivary protein concentrations was assessed. A randomised, controlled, crossover study examined the effects on the salivary proteome of isocaloric doses of various long chain fatty acid (LCFA) oil emulsions compared to no treatment (NT). Fasted males provided saliva samples before and following NT or dosing with LCFA emulsions. The oil component of the DHA emulsion contained predominantly docosahexaenoic acid and the oil component of OA contained predominantly oleic acid. Several proteins were present in significantly (p<0.05) different quantities in saliva samples taken following treatments compared to fasting samples. DHA caused alterations in thioredoxin and serpin B4 relative to OA and NT. A further study evaluated energy intake (EI) in response to LCFA in conjunction with subjective appetite scoring. DHA was associated with significantly lower EI relative to NT and OA (p=0.039). The collective data suggest investigation of salivary proteome may be of value in appetitive response. This article is part of a Special Issue entitled: Proteomics: The clinical link. Copyright © 2011 Elsevier B.V. All rights reserved.

Diversification of the muscle proteome through alternative splicing.

PubMed

Nakka, Kiran; Ghigna, Claudia; Gabellini, Davide; Dilworth, F Jeffrey

2018-03-06

Skeletal muscles express a highly specialized proteome that allows the metabolism of energy sources to mediate myofiber contraction. This muscle-specific proteome is partially derived through the muscle-specific transcription of a subset of genes. Surprisingly, RNA sequencing technologies have also revealed a significant role for muscle-specific alternative splicing in generating protein isoforms that give specialized function to the muscle proteome. In this review, we discuss the current knowledge with respect to the mechanisms that allow pre-mRNA transcripts to undergo muscle-specific alternative splicing while identifying some of the key trans-acting splicing factors essential to the process. The importance of specific splicing events to specialized muscle function is presented along with examples in which dysregulated splicing contributes to myopathies. Though there is now an appreciation that alternative splicing is a major contributor to proteome diversification, the emergence of improved "targeted" proteomic methodologies for detection of specific protein isoforms will soon allow us to better appreciate the extent to which alternative splicing modifies the activity of proteins (and their ability to interact with other proteins) in the skeletal muscle. In addition, we highlight a continued need to better explore the signaling pathways that contribute to the temporal control of trans-acting splicing factor activity to ensure specific protein isoforms are expressed in the proper cellular context. An understanding of the signal-dependent and signal-independent events driving muscle-specific alternative splicing has the potential to provide us with novel therapeutic strategies to treat different myopathies.

A comparative proteomic characterization and nutritional assessment of naturally- and artificially-cultivated Cordyceps sinensis.

PubMed

Zhang, Xu; Liu, Qun; Zhou, Wei; Li, Ping; Alolga, Raphael N; Qi, Lian-Wen; Yin, Xiaojian

2018-06-15

Cordyceps sinensis has gained increasing attention due to its nutritional and medicinal properties. Herein, we employed label-free quantitative mass spectrometry to explore the proteome differences between naturally- and artificially-cultivated C. sinensis. A total of 22,829 peptides with confidence ≥95%, corresponding to 2541 protein groups were identified from the caterpillar bodies/stromata of 12 naturally- and artificially-cultivated samples of C. sinensis. Among them, 165 proteins showed significant differences between the samples of natural and artificial cultivation. These proteins were mainly involved in energy production/conversion, amino acid transport/metabolism, and transcription regulation. The proteomic results were confirmed by the identification of 4 significantly changed metabolites, thus, lysine, threonine, serine, and arginine via untargeted metabolomics. The change tendencies of these metabolites were partly in accordance with changes in abundance of the proteins, which was upstream of their synthetic pathways. In addition, the nutritional value in terms of the levels of nucleosides, nucleotides, and adenosine between the artificially- and naturally-cultivated samples was virtually same. These proteomic data will be useful for understanding the medicinal value of C. sinensis and serve as reference for its artificial cultivation. C. sinensis is a precious and valued medicinal product, the current basic proteome dataset would provide useful information to understand its development/infection processes as well as help to artificially cultivate it. This work would also provide basic proteome profile for further study of C. sinensis. Copyright © 2018. Published by Elsevier B.V.

Mass Spectrometry-Based Proteomics for Pre-Eclampsia and Preterm Birth

PubMed Central

Law, Kai P.; Han, Ting-Li; Tong, Chao; Baker, Philip N.

2015-01-01

Pregnancy-related complications such as pre-eclampsia and preterm birth now represent a notable burden of adverse health. Pre-eclampsia is a hypertensive disorder unique to pregnancy. It is an important cause of maternal death worldwide and a leading cause of fetal growth restriction and iatrogenic prematurity. Fifteen million infants are born preterm each year globally, but more than one million of those do not survive their first month of life. Currently there are no predictive tests available for diagnosis of these pregnancy-related complications and the biological mechanisms of the diseases have not been fully elucidated. Mass spectrometry-based proteomics have all the necessary attributes to provide the needed breakthrough in understanding the pathophysiology of complex human diseases thorough the discovery of biomarkers. The mass spectrometry methodologies employed in the studies for pregnancy-related complications are evaluated in this article. Top-down proteomic and peptidomic profiling by laser mass spectrometry, liquid chromatography or capillary electrophoresis coupled to mass spectrometry, and bottom-up quantitative proteomics and targeted proteomics by liquid chromatography mass spectrometry have been applied to elucidate protein biomarkers and biological mechanism of pregnancy-related complications. The proteomes of serum, urine, amniotic fluid, cervical-vaginal fluid, placental tissue, and cytotrophoblastic cells have all been investigated. Numerous biomarkers or biomarker candidates that could distinguish complicated pregnancies from healthy controls have been proposed. Nevertheless, questions as to the clinically utility and the capacity to elucidate the pathogenesis of the pre-eclampsia and preterm birth remain to be answered. PMID:26006232

Species and tissues specific differentiation of processed animal proteins in aquafeeds using proteomics tools.

PubMed

Rasinger, J D; Marbaix, H; Dieu, M; Fumière, O; Mauro, S; Palmblad, M; Raes, M; Berntssen, M H G

2016-09-16

The rapidly growing aquaculture industry drives the search for sustainable protein sources in fish feed. In the European Union (EU) since 2013 non-ruminant processed animal proteins (PAP) are again permitted to be used in aquafeeds. To ensure that commercial fish feeds do not contain PAP from prohibited species, EU reference methods were established. However, due to the heterogeneous and complex nature of PAP complementary methods are required to guarantee the safe use of this fish feed ingredient. In addition, there is a need for tissue specific PAP detection to identify the sources (i.e. bovine carcass, blood, or meat) of illegal PAP use. In the present study, we investigated and compared different protein extraction, solubilisation and digestion protocols on different proteomics platforms for the detection and differentiation of prohibited PAP. In addition, we assessed if tissue specific PAP detection was feasible using proteomics tools. All work was performed independently in two different laboratories. We found that irrespective of sample preparation gel-based proteomics tools were inappropriate when working with PAP. Gel-free shotgun proteomics approaches in combination with direct spectral comparison were able to provide quality species and tissue specific data to complement and refine current methods of PAP detection and identification. To guarantee the safe use of processed animal protein (PAP) in aquafeeds efficient PAP detection and monitoring tools are required. The present study investigated and compared various proteomics workflows and shows that the application of shotgun proteomics in combination with direct comparison of spectral libraries provides for the desired species and tissue specific classification of this heat sterilized and pressure treated (≥133°C, at 3bar for 20min) protein feed ingredient. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of Missing Proteins Defined by Chromosome-Centric Proteome Project in the Cytoplasmic Detergent-Insoluble Proteins.

PubMed

Chen, Yang; Li, Yaxing; Zhong, Jiayong; Zhang, Jing; Chen, Zhipeng; Yang, Lijuan; Cao, Xin; He, Qing-Yu; Zhang, Gong; Wang, Tong

2015-09-04

Finding protein evidence (PE) for protein coding genes is a primary task of the Phase I Chromosome-Centric Human Proteome Project (C-HPP). Currently, there are 2948 PE level 2-4 coding genes per neXtProt, which are deemed missing proteins in the human proteome. As most samples prepared and analyzed in the C-HPP framework were focusing on detergent soluble proteins, we posit that as a natural composition the cytoplasmic detergent-insoluble proteins (DIPs) represent a source of finding missing proteins. We optimized a workflow and separated cytoplasmic DIPs from three human lung and three human hepatoma cell lines via differential speed centrifugation. We verified that the detergent-soluble proteins (DSPs) could be sufficiently depleted and the cytoplasmic DIP isolation was partially reproducible with Spearman r > 0.70 according to two independent SILAC MS experiments. Through label-free MS, we identified 4524 and 4156 DIPs from lung and liver cells, respectively. Among them, a total of 23 missing proteins (22 PE2 and 1 PE4) were identified by MS, and 18 of them had translation evidence; in addition, six PE5 proteins were identified by MS, three with translation evidence. We showed that cytoplasmic DIPs were not an enrichment of transmembrane proteins and were chromosome-, cell type-, and tissue-specific. Furthermore, we demonstrated that DIPs were distinct from DSPs in terms of structural and physical-chemical features. In conclusion, we have found 23 missing proteins and 6 PE5 proteins from the cytoplasmic insoluble proteome that is biologically and physical-chemically different from the soluble proteome, suggesting that cytoplasmic DIPs carry comprehensive and valuable information for finding PE of missing proteins. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001694.

Prioritization of potential drug targets against P. aeruginosa by core proteomic analysis using computational subtractive genomics and Protein-Protein interaction network.

PubMed

Uddin, Reaz; Jamil, Faiza

2018-06-01

Pseudomonas aeruginosa is an opportunistic gram-negative bacterium that has the capability to acquire resistance under hostile conditions and become a threat worldwide. It is involved in nosocomial infections. In the current study, potential novel drug targets against P. aeruginosa have been identified using core proteomic analysis and Protein-Protein Interactions (PPIs) studies. The non-redundant reference proteome of 68 strains having complete genome and latest assembly version of P. aeruginosa were downloaded from ftp NCBI RefSeq server in October 2016. The standalone CD-HIT tool was used to cluster ortholog proteins (having >=80% amino acid identity) present in all strains. The pan-proteome was clustered in 12,380 Clusters of Orthologous Proteins (COPs). By using in-house shell scripts, 3252 common COPs were extracted out and designated as clusters of core proteome. The core proteome of PAO1 strain was selected by fetching PAO1's proteome from common COPs. As a result, 1212 proteins were shortlisted that are non-homologous to the human but essential for the survival of the pathogen. Among these 1212 proteins, 321 proteins are conserved hypothetical proteins. Considering their potential as drug target, those 321 hypothetical proteins were selected and their probable functions were characterized. Based on the druggability criteria, 18 proteins were shortlisted. The interacting partners were identified by investigating the PPIs network using STRING v10 database. Subsequently, 8 proteins were shortlisted as 'hub proteins' and proposed as potential novel drug targets against P. aeruginosa. The study is interesting for the scientific community working to identify novel drug targets against MDR pathogens particularly P. aeruginosa. Copyright © 2018 Elsevier Ltd. All rights reserved.

Detailed tail proteomic analysis of axolotl (Ambystoma mexicanum) using an mRNA-seq reference database.

PubMed

Demircan, Turan; Keskin, Ilknur; Dumlu, Seda Nilgün; Aytürk, Nilüfer; Avşaroğlu, Mahmut Erhan; Akgün, Emel; Öztürk, Gürkan; Baykal, Ahmet Tarık

2017-01-01

Salamander axolotl has been emerging as an important model for stem cell research due to its powerful regenerative capacity. Several advantages, such as the high capability of advanced tissue, organ, and appendages regeneration, promote axolotl as an ideal model system to extend our current understanding on the mechanisms of regeneration. Acknowledging the common molecular pathways between amphibians and mammals, there is a great potential to translate the messages from axolotl research to mammalian studies. However, the utilization of axolotl is hindered due to the lack of reference databases of genomic, transcriptomic, and proteomic data. Here, we introduce the proteome analysis of the axolotl tail section searched against an mRNA-seq database. We translated axolotl mRNA sequences to protein sequences and annotated these to process the LC-MS/MS data and identified 1001 nonredundant proteins. Functional classification of identified proteins was performed by gene ontology searches. The presence of some of the identified proteins was validated by in situ antibody labeling. Furthermore, we have analyzed the proteome expressional changes postamputation at three time points to evaluate the underlying mechanisms of the regeneration process. Taken together, this work expands the proteomics data of axolotl to contribute to its establishment as a fully utilized model. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Golgi Enrichment and Proteomic Analysis of Developing Pinus radiata Xylem by Free-Flow Electrophoresis

PubMed Central

Macdonald, Lucy J.; Adams, Paul D.; Petzold, Christopher J.; Strabala, Timothy J.; Wagner, Armin; Heazlewood, Joshua L.

2013-01-01

Our understanding of the contribution of Golgi proteins to cell wall and wood formation in any woody plant species is limited. Currently, little Golgi proteomics data exists for wood-forming tissues. In this study, we attempted to address this issue by generating and analyzing Golgi-enriched membrane preparations from developing xylem of compression wood from the conifer Pinus radiata. Developing xylem samples from 3-year-old pine trees were harvested for this purpose at a time of active growth and subjected to a combination of density centrifugation followed by free flow electrophoresis, a surface charge separation technique used in the enrichment of Golgi membranes. This combination of techniques was successful in achieving an approximately 200-fold increase in the activity of the Golgi marker galactan synthase and represents a significant improvement for proteomic analyses of the Golgi from conifers. A total of thirty known Golgi proteins were identified by mass spectrometry including glycosyltransferases from gene families involved in glucomannan and glucuronoxylan biosynthesis. The free flow electrophoresis fractions of enriched Golgi were highly abundant in structural proteins (actin and tubulin) indicating a role for the cytoskeleton during compression wood formation. The mass spectrometry proteomics data associated with this study have been deposited to the ProteomeXchange with identifier PXD000557. PMID:24416096

Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human.

PubMed

Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

2016-04-13

Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish.

Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human

PubMed Central

Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

2016-01-01

Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish. PMID:27071722

A Statistical Selection Strategy for Normalization Procedures in LC-MS Proteomics Experiments through Dataset Dependent Ranking of Normalization Scaling Factors

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

Webb-Robertson, Bobbie-Jo M.; Matzke, Melissa M.; Jacobs, Jon M.

2011-12-01

Quantification of LC-MS peak intensities assigned during peptide identification in a typical comparative proteomics experiment will deviate from run-to-run of the instrument due to both technical and biological variation. Thus, normalization of peak intensities across a LC-MS proteomics dataset is a fundamental step in pre-processing. However, the downstream analysis of LC-MS proteomics data can be dramatically affected by the normalization method selected . Current normalization procedures for LC-MS proteomics data are presented in the context of normalization values derived from subsets of the full collection of identified peptides. The distribution of these normalization values is unknown a priori. If theymore » are not independent from the biological factors associated with the experiment the normalization process can introduce bias into the data, which will affect downstream statistical biomarker discovery. We present a novel approach to evaluate normalization strategies, where a normalization strategy includes the peptide selection component associated with the derivation of normalization values. Our approach evaluates the effect of normalization on the between-group variance structure in order to identify candidate normalization strategies that improve the structure of the data without introducing bias into the normalized peak intensities.« less

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

Data-Driven Approach To Determine Popular Proteins for Targeted Proteomics Translation of Six Organ Systems.

PubMed

Lam, Maggie P Y; Venkatraman, Vidya; Xing, Yi; Lau, Edward; Cao, Quan; Ng, Dominic C M; Su, Andrew I; Ge, Junbo; Van Eyk, Jennifer E; Ping, Peipei

2016-11-04

Amidst the proteomes of human tissues lie subsets of proteins that are closely involved in conserved pathophysiological processes. Much of biomedical research concerns interrogating disease signature proteins and defining their roles in disease mechanisms. With advances in proteomics technologies, it is now feasible to develop targeted proteomics assays that can accurately quantify protein abundance as well as their post-translational modifications; however, with rapidly accumulating number of studies implicating proteins in diseases, current resources are insufficient to target every protein without judiciously prioritizing the proteins with high significance and impact for assay development. We describe here a data science method to prioritize and expedite assay development on high-impact proteins across research fields by leveraging the biomedical literature record to rank and normalize proteins that are popularly and preferentially published by biomedical researchers. We demonstrate this method by finding priority proteins across six major physiological systems (cardiovascular, cerebral, hepatic, renal, pulmonary, and intestinal). The described method is data-driven and builds upon the collective knowledge of previous publications referenced on PubMed to lend objectivity to target selection. The method and resulting popular protein lists may also be useful for exploring biological processes associated with various physiological systems and research topics, in addition to benefiting ongoing efforts to facilitate the broad translation of proteomics technologies.

In-Depth Characterization of Sheep (Ovis aries) Milk Whey Proteome and Comparison with Cow (Bos taurus)

PubMed Central

Ha, Minh; Sabherwal, Manya; Duncan, Elizabeth; Stevens, Stewart; Stockwell, Peter; McConnell, Michelle; Bekhit, Alaa El-Din; Carne, Alan

2015-01-01

An in-depth proteomic study of sheep milk whey is reported and compared to the data available in the literature for the cow whey proteome. A combinatorial peptide ligand library kit (ProteoMiner) was used to normalize protein abundance in the sheep whey proteome followed by an in-gel digest of a 1D-PAGE display and an in-solution digestion followed by OFFGEL isoelectric focusing fractionation. The peptide fractions obtained were then analyzed by LC-MS/MS. This enabled identification of 669 proteins in sheep whey that, to our knowledge, is the largest inventory of sheep whey proteins identified to date. A comprehensive list of cow whey proteins currently available in the literature (783 proteins from unique genes) was assembled and compared to the sheep whey proteome data obtained in this study (606 proteins from unique genes). This comparison revealed that while the 233 proteins shared by the two species were significantly enriched for immune and inflammatory responses in gene ontology analysis, proteins only found in sheep whey in this study were identified that take part in both cellular development and immune responses, whereas proteins only found in cow whey in this study were identified to be associated with metabolism and cellular growth. PMID:26447763

Current understanding of the human microbiome.

PubMed

Gilbert, Jack A; Blaser, Martin J; Caporaso, J Gregory; Jansson, Janet K; Lynch, Susan V; Knight, Rob

2018-04-10

Our understanding of the link between the human microbiome and disease, including obesity, inflammatory bowel disease, arthritis and autism, is rapidly expanding. Improvements in the throughput and accuracy of DNA sequencing of the genomes of microbial communities that are associated with human samples, complemented by analysis of transcriptomes, proteomes, metabolomes and immunomes and by mechanistic experiments in model systems, have vastly improved our ability to understand the structure and function of the microbiome in both diseased and healthy states. However, many challenges remain. In this review, we focus on studies in humans to describe these challenges and propose strategies that leverage existing knowledge to move rapidly from correlation to causation and ultimately to translation into therapies.

Exploration of the medical periodic table: towards new targets.

PubMed

Barry, Nicolas P E; Sadler, Peter J

2013-06-07

Metallodrugs offer potential for unique mechanisms of drug action based on the choice of the metal, its oxidation state, the types and number of coordinated ligands and the coordination geometry. We discuss recent progress in identifying new target sites and elucidating the mechanisms of action of anti-cancer, anti-bacterial, anti-viral, anti-parasitic, anti-inflammatory, and anti-neurodegenerative agents, as well as in the design of metal-based diagnostic agents. Progress in identifying and defining target sites has been accelerated recently by advances in proteomics, genomics and metal speciation analysis. Examples of metal compounds and chelating agents (enzyme inhibitors) currently in clinical use, clinical trials or preclinical development are highlighted.

Current understanding of the human microbiome

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

Gilbert, Jack A.; Blaser, Martin J.; Caporaso, J. Gregory

Our understanding of the link between the human microbiome and disease, including obesity, inflammatory bowel disease, arthritis and autism, is rapidly expanding. Improvements in the throughput and accuracy of DNA sequencing of the genomes of microbial communities associated with human samples, complemented by analysis of transcriptomes, proteomes, metabolomes and immunomes, and mechanistic experiments in model systems, have vastly improved our ability to understand the structure and function of the microbiome in both diseased and healthy states. However, many challenges remain. In this Review we focus on studies in humans to describe these challenges, and propose strategies that leverage existing knowledgemore » to move rapidly from correlation to causation, and ultimately to translation.« less

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

NCI Releases Video: Proteogenomics Research - On the Frontier of Precision Medicine | Office of Cancer Clinical Proteomics Research

Cancer.gov

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI), part of the National Institutes of Health, announces the release of an educational video titled “Proteogenomics Research: On the Frontier of Precision Medicine."  Launched at the HUPO2017 Global Leadership Gala Dinner, catalyzed in part by the Cancer Moonshot initiative and featuring as keynote speaker the 47th Vice President of the United States of America Joseph R.

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

Ultraweak photon emission and proteomics analyses in soybean under abiotic stress.

PubMed

Komatsu, Setsuko; Kamal, Abu Hena Mostafa; Makino, Takahiro; Hossain, Zahed

2014-07-01

Biophotons are ultraweak photon emissions that are closely related to various biological activities and processes. In mammals, biophoton emissions originate from oxidative bursts in immunocytes during immunological responses. Biophotons emitted from plant organs provide novel information about the physiological state of plant under in vivo condition. In this review, the principles and recent advances in the measurement of biophoton emissions in plants are described. Furthermore, examples of biophoton emission and proteomics in soybean under abiotic stress are reviewed and discussed. Finally, this review suggests that the application of proteomics should provide a better interpretation of plant response to biophoton emission and allow the identification of genes that will allow the screening of crops able to produce maximal yields, even in stressful environments. Copyright © 2014 Elsevier B.V. All rights reserved.

A perspective on extracellular vesicles proteomics

NASA Astrophysics Data System (ADS)

Rosa-Fernandes, Livia; Rocha, Victória Bombarda; Carregari, Victor Corasolla; Urbani, Andrea; Palmisano, Giuseppe

2017-11-01

Increasing attention has been given to secreted extracellular vesicles (EVs) in the past decades, especially in the portrayal of their molecular cargo and role as messengers in both homeostasis and pathophysiological conditions. This review presents the state-of-the-art proteomic technologies to identify and quantify EVs proteins along with their PTMs, interacting partners and structural details. The rapid growth of mass spectrometry-based analytical strategies for protein sequencing, PTMs and structural characterization has improved the level of molecular details that can be achieve from limited amount of EVs isolated from different biological sources. Here we will provide a perspective view on the achievements and challenges on EVs proteome characterization using mass spectrometry. A detailed bioinformatics approach will help us to picture the molecular fingerprint of EVs and understand better their pathophysiological function.

The Resistome: A Comprehensive Database of Escherichia coli Resistance Phenotypes.

PubMed

Winkler, James D; Halweg-Edwards, Andrea L; Erickson, Keesha E; Choudhury, Alaksh; Pines, Gur; Gill, Ryan T

2016-12-16

The microbial ability to resist stressful environmental conditions and chemical inhibitors is of great industrial and medical interest. Much of the data related to mutation-based stress resistance, however, is scattered through the academic literature, making it difficult to apply systematic analyses to this wealth of information. To address this issue, we introduce the Resistome database: a literature-curated collection of Escherichia coli genotypes-phenotypes containing over 5,000 mutants that resist hundreds of compounds and environmental conditions. We use the Resistome to understand our current state of knowledge regarding resistance and to detect potential synergy or antagonism between resistance phenotypes. Our data set represents one of the most comprehensive collections of genomic data related to resistance currently available. Future development will focus on the construction of a combined genomic-transcriptomic-proteomic framework for understanding E. coli's resistance biology. The Resistome can be downloaded at https://bitbucket.org/jdwinkler/resistome_release/overview .

Inconsistencies in the red blood cell membrane proteome analysis: generation of a database for research and diagnostic applications

PubMed Central

Hegedűs, Tamás; Chaubey, Pururawa Mayank; Várady, György; Szabó, Edit; Sarankó, Hajnalka; Hofstetter, Lia; Roschitzki, Bernd; Sarkadi, Balázs

2015-01-01

Based on recent results, the determination of the easily accessible red blood cell (RBC) membrane proteins may provide new diagnostic possibilities for assessing mutations, polymorphisms or regulatory alterations in diseases. However, the analysis of the current mass spectrometry-based proteomics datasets and other major databases indicates inconsistencies—the results show large scattering and only a limited overlap for the identified RBC membrane proteins. Here, we applied membrane-specific proteomics studies in human RBC, compared these results with the data in the literature, and generated a comprehensive and expandable database using all available data sources. The integrated web database now refers to proteomic, genetic and medical databases as well, and contains an unexpected large number of validated membrane proteins previously thought to be specific for other tissues and/or related to major human diseases. Since the determination of protein expression in RBC provides a method to indicate pathological alterations, our database should facilitate the development of RBC membrane biomarker platforms and provide a unique resource to aid related further research and diagnostics. Database URL: http://rbcc.hegelab.org PMID:26078478

Using Proteomics to Understand How Leishmania Parasites Survive inside the Host and Establish Infection

PubMed Central

Veras, Patrícia Sampaio Tavares; Bezerra de Menezes, Juliana Perrone

2016-01-01

Leishmania is a protozoan parasite that causes a wide range of different clinical manifestations in mammalian hosts. It is a major public health risk on different continents and represents one of the most important neglected diseases. Due to the high toxicity of the drugs currently used, and in the light of increasing drug resistance, there is a critical need to develop new drugs and vaccines to control Leishmania infection. Over the past few years, proteomics has become an important tool to understand the underlying biology of Leishmania parasites and host interaction. The large-scale study of proteins, both in parasites and within the host in response to infection, can accelerate the discovery of new therapeutic targets. By studying the proteomes of host cells and tissues infected with Leishmania, as well as changes in protein profiles among promastigotes and amastigotes, scientists hope to better understand the biology involved in the parasite survival and the host-parasite interaction. This review demonstrates the feasibility of proteomics as an approach to identify new proteins involved in Leishmania differentiation and intracellular survival. PMID:27548150

Using Proteomics to Understand How Leishmania Parasites Survive inside the Host and Establish Infection.

PubMed

Veras, Patrícia Sampaio Tavares; Bezerra de Menezes, Juliana Perrone

2016-08-19

Leishmania is a protozoan parasite that causes a wide range of different clinical manifestations in mammalian hosts. It is a major public health risk on different continents and represents one of the most important neglected diseases. Due to the high toxicity of the drugs currently used, and in the light of increasing drug resistance, there is a critical need to develop new drugs and vaccines to control Leishmania infection. Over the past few years, proteomics has become an important tool to understand the underlying biology of Leishmania parasites and host interaction. The large-scale study of proteins, both in parasites and within the host in response to infection, can accelerate the discovery of new therapeutic targets. By studying the proteomes of host cells and tissues infected with Leishmania, as well as changes in protein profiles among promastigotes and amastigotes, scientists hope to better understand the biology involved in the parasite survival and the host-parasite interaction. This review demonstrates the feasibility of proteomics as an approach to identify new proteins involved in Leishmania differentiation and intracellular survival.

Computational approaches to protein inference in shotgun proteomics

PubMed Central

2012-01-01

Shotgun proteomics has recently emerged as a powerful approach to characterizing proteomes in biological samples. Its overall objective is to identify the form and quantity of each protein in a high-throughput manner by coupling liquid chromatography with tandem mass spectrometry. As a consequence of its high throughput nature, shotgun proteomics faces challenges with respect to the analysis and interpretation of experimental data. Among such challenges, the identification of proteins present in a sample has been recognized as an important computational task. This task generally consists of (1) assigning experimental tandem mass spectra to peptides derived from a protein database, and (2) mapping assigned peptides to proteins and quantifying the confidence of identified proteins. Protein identification is fundamentally a statistical inference problem with a number of methods proposed to address its challenges. In this review we categorize current approaches into rule-based, combinatorial optimization and probabilistic inference techniques, and present them using integer programing and Bayesian inference frameworks. We also discuss the main challenges of protein identification and propose potential solutions with the goal of spurring innovative research in this area. PMID:23176300

Short communication: Proteomic characterization of tuberculin purified protein derivative from Mycobacterium bovis.

PubMed

Cho, Yun Sang; Jang, Young-Boo; Lee, Sang-Eun; Cho, Je-Yoel; Ahn, Jung-Mo; Hwang, Inyeong; Heo, Eunjeong; Nam, Hyang-Mi; Cho, Donghee; Her, Moon; Jean, Young Hwa; Jung, Suk Chan; Kim, Jong Man; Lee, Hee Soo; Lee, Keechan; Belisle, John T

2015-08-01

Bovine tuberculin purified protein derivative (bPPD) is used as an intradermal test (IT) reagent to detect bovine tuberculosis (bTB) in most countries. Identification of bPPD proteins is critical to understanding the immunological reaction of IT at the molecular level. While bPPD from the United Kingdom (UK) and Brazil (BR) have been recently defined at the proteomic level, bPPD from the Republic of Korea (KR) has not yet been analyzed. Here, bPPD KR proteome was examined for the first time. In total, 271 proteins were identified, including Mycobacterium bovis-specific proteins Mb0854c and Mb2898, and 42 known T cell antigens. On comparing with proteomes of bPPD UK and BR, 33 proteins were found to be common among all three bPPDs, of which 15 proteins were T cell antigens. M. bovis-specific antigens with T cell activity in bPPD may be novel candidates for use as alternatives to currently available bPPD in diagnostics. Copyright © 2015 Elsevier Ltd. All rights reserved.

ProGeRF: Proteome and Genome Repeat Finder Utilizing a Fast Parallel Hash Function

PubMed Central

Moraes, Walas Jhony Lopes; Rodrigues, Thiago de Souza; Bartholomeu, Daniella Castanheira

2015-01-01

Repetitive element sequences are adjacent, repeating patterns, also called motifs, and can be of different lengths; repetitions can involve their exact or approximate copies. They have been widely used as molecular markers in population biology. Given the sizes of sequenced genomes, various bioinformatics tools have been developed for the extraction of repetitive elements from DNA sequences. However, currently available tools do not provide options for identifying repetitive elements in the genome or proteome, displaying a user-friendly web interface, and performing-exhaustive searches. ProGeRF is a web site for extracting repetitive regions from genome and proteome sequences. It was designed to be efficient, fast, and accurate and primarily user-friendly web tool allowing many ways to view and analyse the results. ProGeRF (Proteome and Genome Repeat Finder) is freely available as a stand-alone program, from which the users can download the source code, and as a web tool. It was developed using the hash table approach to extract perfect and imperfect repetitive regions in a (multi)FASTA file, while allowing a linear time complexity. PMID:25811026

OralCard: a bioinformatic tool for the study of oral proteome.

PubMed

Arrais, Joel P; Rosa, Nuno; Melo, José; Coelho, Edgar D; Amaral, Diana; Correia, Maria José; Barros, Marlene; Oliveira, José Luís

2013-07-01

The molecular complexity of the human oral cavity can only be clarified through identification of components that participate within it. However current proteomic techniques produce high volumes of information that are dispersed over several online databases. Collecting all of this data and using an integrative approach capable of identifying unknown associations is still an unsolved problem. This is the main motivation for this work. We present the online bioinformatic tool OralCard, which comprises results from 55 manually curated articles reflecting the oral molecular ecosystem (OralPhysiOme). It comprises experimental information available from the oral proteome both of human (OralOme) and microbial origin (MicroOralOme) structured in protein, disease and organism. This tool is a key resource for researchers to understand the molecular foundations implicated in biology and disease mechanisms of the oral cavity. The usefulness of this tool is illustrated with the analysis of the oral proteome associated with diabetes melitus type 2. OralCard is available at http://bioinformatics.ua.pt/oralcard. Copyright © 2013 Elsevier Ltd. All rights reserved.

Proteomics of the Lysosome

PubMed Central

Lübke, Torben; Lobel, Peter; Sleat, David

2009-01-01

Defects in lysosomal function have been associated with numerous monogenic human diseases typically classified as lysosomal storage diseases. However, there is increasing evidence that lysosomal proteins are also involved in more widespread human diseases including cancer and Alzheimer disease. Thus, there is a continuing interest in understanding the cellular functions of the lysosome and an emerging approach to this is the identification of its constituent proteins by proteomic analyses. To date, the mammalian lysosome has been shown to contain ~ 60 soluble luminal proteins and ~25 transmembrane proteins. However, recent proteomic studies based upon affinity purification of soluble components or subcellular fractionation to obtain both soluble and membrane components suggest that there may be many more of both classes of protein resident within this organelle than previously appreciated. Discovery of such proteins has important implications for understanding the function and the dynamics of the lysosome but can also lead the way towards the discovery of the genetic basis for human diseases of hitherto unknown etiology. Here, we describe current approaches to lysosomal proteomics and data interpretation and review the new lysosomal proteins that have recently emerged from such studies. PMID:18977398

Computer aided manual validation of mass spectrometry-based proteomic data.

PubMed

Curran, Timothy G; Bryson, Bryan D; Reigelhaupt, Michael; Johnson, Hannah; White, Forest M

2013-06-15

Advances in mass spectrometry-based proteomic technologies have increased the speed of analysis and the depth provided by a single analysis. Computational tools to evaluate the accuracy of peptide identifications from these high-throughput analyses have not kept pace with technological advances; currently the most common quality evaluation methods are based on statistical analysis of the likelihood of false positive identifications in large-scale data sets. While helpful, these calculations do not consider the accuracy of each identification, thus creating a precarious situation for biologists relying on the data to inform experimental design. Manual validation is the gold standard approach to confirm accuracy of database identifications, but is extremely time-intensive. To palliate the increasing time required to manually validate large proteomic datasets, we provide computer aided manual validation software (CAMV) to expedite the process. Relevant spectra are collected, catalogued, and pre-labeled, allowing users to efficiently judge the quality of each identification and summarize applicable quantitative information. CAMV significantly reduces the burden associated with manual validation and will hopefully encourage broader adoption of manual validation in mass spectrometry-based proteomics. Copyright © 2013 Elsevier Inc. All rights reserved.

iAB-RBC-283: A proteomically derived knowledge-base of erythrocyte metabolism that can be used to simulate its physiological and patho-physiological states.

PubMed

Bordbar, Aarash; Jamshidi, Neema; Palsson, Bernhard O

2011-07-12

The development of high-throughput technologies capable of whole cell measurements of genes, proteins, and metabolites has led to the emergence of systems biology. Integrated analysis of the resulting omic data sets has proved to be hard to achieve. Metabolic network reconstructions enable complex relationships amongst molecular components to be represented formally in a biologically relevant manner while respecting physical constraints. In silico models derived from such reconstructions can then be queried or interrogated through mathematical simulations. Proteomic profiling studies of the mature human erythrocyte have shown more proteins present related to metabolic function than previously thought; however the significance and the causal consequences of these findings have not been explored. Erythrocyte proteomic data was used to reconstruct the most expansive description of erythrocyte metabolism to date, following extensive manual curation, assessment of the literature, and functional testing. The reconstruction contains 281 enzymes representing functions from glycolysis to cofactor and amino acid metabolism. Such a comprehensive view of erythrocyte metabolism implicates the erythrocyte as a potential biomarker for different diseases as well as a 'cell-based' drug-screening tool. The analysis shows that 94 erythrocyte enzymes are implicated in morbid single nucleotide polymorphisms, representing 142 pathologies. In addition, over 230 FDA-approved and experimental pharmaceuticals have enzymatic targets in the erythrocyte. The advancement of proteomic technologies and increased generation of high-throughput proteomic data have created the need for a means to analyze these data in a coherent manner. Network reconstructions provide a systematic means to integrate and analyze proteomic data in a biologically meaning manner. Analysis of the red cell proteome has revealed an unexpected level of complexity in the functional capabilities of human erythrocyte metabolism.

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/.

Accounting for the Multiple Natures of Missing Values in Label-Free Quantitative Proteomics Data Sets to Compare Imputation Strategies.

PubMed

Lazar, Cosmin; Gatto, Laurent; Ferro, Myriam; Bruley, Christophe; Burger, Thomas

2016-04-01

Missing values are a genuine issue in label-free quantitative proteomics. Recent works have surveyed the different statistical methods to conduct imputation and have compared them on real or simulated data sets and recommended a list of missing value imputation methods for proteomics application. Although insightful, these comparisons do not account for two important facts: (i) depending on the proteomics data set, the missingness mechanism may be of different natures and (ii) each imputation method is devoted to a specific type of missingness mechanism. As a result, we believe that the question at stake is not to find the most accurate imputation method in general but instead the most appropriate one. We describe a series of comparisons that support our views: For instance, we show that a supposedly "under-performing" method (i.e., giving baseline average results), if applied at the "appropriate" time in the data-processing pipeline (before or after peptide aggregation) on a data set with the "appropriate" nature of missing values, can outperform a blindly applied, supposedly "better-performing" method (i.e., the reference method from the state-of-the-art). This leads us to formulate few practical guidelines regarding the choice and the application of an imputation method in a proteomics context.

Current algorithmic solutions for peptide-based proteomics data generation and identification.

PubMed

Hoopmann, Michael R; Moritz, Robert L

2013-02-01

Peptide-based proteomic data sets are ever increasing in size and complexity. These data sets provide computational challenges when attempting to quickly analyze spectra and obtain correct protein identifications. Database search and de novo algorithms must consider high-resolution MS/MS spectra and alternative fragmentation methods. Protein inference is a tricky problem when analyzing large data sets of degenerate peptide identifications. Combining multiple algorithms for improved peptide identification puts significant strain on computational systems when investigating large data sets. This review highlights some of the recent developments in peptide and protein identification algorithms for analyzing shotgun mass spectrometry data when encountering the aforementioned hurdles. Also explored are the roles that analytical pipelines, public spectral libraries, and cloud computing play in the evolution of peptide-based proteomics. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

Kolker, Eugene

Our project focused primarily on analysis of different types of data produced by global high-throughput technologies, data integration of gene annotation, and gene and protein expression information, as well as on getting a better functional annotation of Shewanella genes. Specifically, four of our numerous major activities and achievements include the development of: statistical models for identification and expression proteomics, superior to currently available approaches (including our own earlier ones); approaches to improve gene annotations on the whole-organism scale; standards for annotation, transcriptomics and proteomics approaches; and generalized approaches for data integration of gene annotation, gene and protein expression information.

The Role of Proteomics in the Diagnosis and Treatment of Women's Cancers: Current Trends in Technology and Future Opportunities

PubMed Central

Breuer, Eun-Kyoung Yim; Murph, Mandi M.

2011-01-01

Technological and scientific innovations over the last decade have greatly contributed to improved diagnostics, predictive models, and prognosis among cancers affecting women. In fact, an explosion of information in these areas has almost assured future generations that outcomes in cancer will continue to improve. Herein we discuss the current status of breast, cervical, and ovarian cancers as it relates to screening, disease diagnosis, and treatment options. Among the differences in these cancers, it is striking that breast cancer has multiple predictive tests based upon tumor biomarkers and sophisticated, individualized options for prescription therapeutics while ovarian cancer lacks these tools. In addition, cervical cancer leads the way in innovative, cancer-preventative vaccines and multiple screening options to prevent disease progression. For each of these malignancies, emerging proteomic technologies based upon mass spectrometry, stable isotope labeling with amino acids, high-throughput ELISA, tissue or protein microarray techniques, and click chemistry in the pursuit of activity-based profiling can pioneer the next generation of discovery. We will discuss six of the latest techniques to understand proteomics in cancer and highlight research utilizing these techniques with the goal of improvement in the management of women's cancers. PMID:21886869

Proteomic analysis of acquired tamoxifen resistance in MCF-7 cells reveals expression signatures associated with enhanced migration

PubMed Central

2012-01-01

Introduction Acquired tamoxifen resistance involves complex signaling events that are not yet fully understood. Successful therapeutic intervention to delay the onset of hormone resistance depends critically on mechanistic elucidation of viable molecular targets associated with hormone resistance. This study was undertaken to investigate the global proteomic alterations in a tamoxifen resistant MCF-7 breast cancer cell line obtained by long term treatment of the wild type MCF-7 cell line with 4-hydroxytamoxifen (4-OH Tam). Methods We cultured MCF-7 cells with 4-OH Tam over a period of 12 months to obtain the resistant cell line. A gel-free, quantitative proteomic method was used to identify and quantify the proteome of the resistant cell line. Nano-flow high-performance liquid chromatography coupled to high resolution Fourier transform mass spectrometry was used to analyze fractionated peptide mixtures that were isobarically labeled from the resistant and control cell lysates. Real time quantitative PCR and Western blots were used to verify selected proteomic changes. Lentiviral vector transduction was used to generate MCF-7 cells stably expressing S100P. Online pathway analysis was performed to assess proteomic signatures in tamoxifen resistance. Survival analysis was done to evaluate clinical relevance of altered proteomic expressions. Results Quantitative proteomic analysis revealed a wide breadth of signaling events during transition to acquired tamoxifen resistance. A total of 629 proteins were found significantly changed with 364 up-regulated and 265 down-regulated. Collectively, these changes demonstrated the suppressed state of estrogen receptor (ER) and ER-regulated genes, activated survival signaling and increased migratory capacity of the resistant cell line. The protein S100P was found to play a critical role in conferring tamoxifen resistance and enhanced cell motility. Conclusions Our data demonstrate that the adaptive changes in the proteome of tamoxifen resistant breast cancer cells are characterized by down-regulated ER signaling, activation of alternative survival pathways, and enhanced cell motility through regulation of the actin cytoskeleton dynamics. Evidence also emerged that S100P mediates acquired tamoxifen resistance and migration capacity. PMID:22417809

Animal board invited review: advances in proteomics for animal and food sciences.

PubMed

Almeida, A M; Bassols, A; Bendixen, E; Bhide, M; Ceciliani, F; Cristobal, S; Eckersall, P D; Hollung, K; Lisacek, F; Mazzucchelli, G; McLaughlin, M; Miller, I; Nally, J E; Plowman, J; Renaut, J; Rodrigues, P; Roncada, P; Staric, J; Turk, R

2015-01-01

Animal production and health (APH) is an important sector in the world economy, representing a large proportion of the budget of all member states in the European Union and in other continents. APH is a highly competitive sector with a strong emphasis on innovation and, albeit with country to country variations, on scientific research. Proteomics (the study of all proteins present in a given tissue or fluid - i.e. the proteome) has an enormous potential when applied to APH. Nevertheless, for a variety of reasons and in contrast to disciplines such as plant sciences or human biomedicine, such potential is only now being tapped. To counter such limited usage, 6 years ago we created a consortium dedicated to the applications of Proteomics to APH, specifically in the form of a Cooperation in Science and Technology (COST) Action, termed FA1002--Proteomics in Farm Animals: www.cost-faproteomics.org. In 4 years, the consortium quickly enlarged to a total of 31 countries in Europe, as well as Israel, Argentina, Australia and New Zealand. This article has a triple purpose. First, we aim to provide clear examples on the applications and benefits of the use of proteomics in all aspects related to APH. Second, we provide insights and possibilities on the new trends and objectives for APH proteomics applications and technologies for the years to come. Finally, we provide an overview and balance of the major activities and accomplishments of the COST Action on Farm Animal Proteomics. These include activities such as the organization of seminars, workshops and major scientific conferences, organization of summer schools, financing Short-Term Scientific Missions (STSMs) and the generation of scientific literature. Overall, the Action has attained all of the proposed objectives and has made considerable difference by putting proteomics on the global map for animal and veterinary researchers in general and by contributing significantly to reduce the East-West and North-South gaps existing in the European farm animal research. Future activities of significance in the field of scientific research, involving members of the action, as well as others, will likely be established in the future.

Analysis of the early heterocyst Cys-proteome in the multicellular cyanobacterium Nostoc punctiforme reveals novel insights into the division of labor within diazotrophic filaments.

PubMed

Sandh, Gustaf; Ramström, Margareta; Stensjö, Karin

2014-12-04

In the filamentous cyanobacterium Nostoc punctiforme ATCC 29133, removal of combined nitrogen induces the differentiation of heterocysts, a cell-type specialized in N2 fixation. The differentiation involves genomic, structural and metabolic adaptations. In cyanobacteria, changes in the availability of carbon and nitrogen have also been linked to redox regulated posttranslational modifications of protein bound thiol groups. We have here employed a thiol targeting strategy to relatively quantify the putative redox proteome in heterocysts as compared to N2-fixing filaments, 24 hours after combined nitrogen depletion. The aim of the study was to expand the coverage of the cell-type specific proteome and metabolic landscape of heterocysts. Here we report the first cell-type specific proteome of newly formed heterocysts, compared to N2-fixing filaments, using the cysteine-specific selective ICAT methodology. The data set defined a good quantitative accuracy of the ICAT reagent in complex protein samples. The relative abundance levels of 511 proteins were determined and 74% showed a cell-type specific differential abundance. The majority of the identified proteins have not previously been quantified at the cell-type specific level. We have in addition analyzed the cell-type specific differential abundance of a large section of proteins quantified in both newly formed and steady-state diazotrophic cultures in N. punctiforme. The results describe a wide distribution of members of the putative redox regulated Cys-proteome in the central metabolism of both vegetative cells and heterocysts of N. punctiforme. The data set broadens our understanding of heterocysts and describes novel proteins involved in heterocyst physiology, including signaling and regulatory proteins as well as a large number of proteins with unknown function. Significant differences in cell-type specific abundance levels were present in the cell-type specific proteomes of newly formed diazotrophic filaments as compared to steady-state cultures. Therefore we conclude that by using our approach we are able to analyze a synchronized fraction of newly formed heterocysts, which enabled a better detection of proteins involved in the heterocyst specific physiology.

Improved prediction of peptide detectability for targeted proteomics using a rank-based algorithm and organism-specific data.

PubMed

Qeli, Ermir; Omasits, Ulrich; Goetze, Sandra; Stekhoven, Daniel J; Frey, Juerg E; Basler, Konrad; Wollscheid, Bernd; Brunner, Erich; Ahrens, Christian H

2014-08-28

The in silico prediction of the best-observable "proteotypic" peptides in mass spectrometry-based workflows is a challenging problem. Being able to accurately predict such peptides would enable the informed selection of proteotypic peptides for targeted quantification of previously observed and non-observed proteins for any organism, with a significant impact for clinical proteomics and systems biology studies. Current prediction algorithms rely on physicochemical parameters in combination with positive and negative training sets to identify those peptide properties that most profoundly affect their general detectability. Here we present PeptideRank, an approach that uses learning to rank algorithm for peptide detectability prediction from shotgun proteomics data, and that eliminates the need to select a negative dataset for the training step. A large number of different peptide properties are used to train ranking models in order to predict a ranking of the best-observable peptides within a protein. Empirical evaluation with rank accuracy metrics showed that PeptideRank complements existing prediction algorithms. Our results indicate that the best performance is achieved when it is trained on organism-specific shotgun proteomics data, and that PeptideRank is most accurate for short to medium-sized and abundant proteins, without any loss in prediction accuracy for the important class of membrane proteins. Targeted proteomics approaches have been gaining a lot of momentum and hold immense potential for systems biology studies and clinical proteomics. However, since only very few complete proteomes have been reported to date, for a considerable fraction of a proteome there is no experimental proteomics evidence that would allow to guide the selection of the best-suited proteotypic peptides (PTPs), i.e. peptides that are specific to a given proteoform and that are repeatedly observed in a mass spectrometer. We describe a novel, rank-based approach for the prediction of the best-suited PTPs for targeted proteomics applications. By building on methods developed in the field of information retrieval (e.g. web search engines like Google's PageRank), we circumvent the delicate step of selecting positive and negative training sets and at the same time also more closely reflect the experimentalist´s need for selecting e.g. the 5 most promising peptides for targeting a protein of interest. This approach allows to predict PTPs for not yet observed proteins or for organisms without prior experimental proteomics data such as many non-model organisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation

PubMed Central

Metcalfe, Clive; Ramasubramoni, Anjana; Pula, Giordano; Harper, Matthew T.; Mundell, Stuart J.; Coxon, Carmen H.

2016-01-01

Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents. PMID:27716777

Selective Targeting of the Cysteine Proteome by Thioredoxin and Glutathione Redox Systems

PubMed Central

Go, Young-Mi; Roede, James R.; Walker, Douglas I.; Duong, Duc M.; Seyfried, Nicholas T.; Orr, Michael; Liang, Yongliang; Pennell, Kurt D.; Jones, Dean P.

2013-01-01

Thioredoxin (Trx) and GSH are the major thiol antioxidants protecting cells from oxidative stress-induced cytotoxicity. Redox states of Trx and GSH have been used as indicators of oxidative stress. Accumulating studies suggest that Trx and GSH redox systems regulate cell signaling and metabolic pathways differently and independently during diverse stressful conditions. In the current study, we used a mass spectrometry-based redox proteomics approach to test responses of the cysteine (Cys) proteome to selective disruption of the Trx- and GSH-dependent systems. Auranofin (ARF) was used to inhibit Trx reductase without detectable oxidation of the GSH/GSSG couple, and buthionine sulfoximine (BSO) was used to deplete GSH without detectable oxidation of Trx1. Results for 606 Cys-containing peptides (peptidyl Cys) showed that 36% were oxidized more than 1.3-fold by ARF, whereas BSO-induced oxidation of peptidyl Cys was only 10%. Mean fold oxidation of these peptides was also higher by ARF than BSO treatment. Analysis of potential functional pathways showed that ARF oxidized peptides associated with glycolysis, cytoskeleton remodeling, translation and cell adhesion. Of 60 peptidyl Cys oxidized due to depletion of GSH, 41 were also oxidized by ARF and included proteins of translation and cell adhesion but not glycolysis or cytoskeletal remodeling. Studies to test functional correlates showed that pyruvate kinase activity and lactate levels were decreased with ARF but not BSO, confirming the effects on glycolysis-associated proteins are sensitive to oxidation by ARF. These data show that the Trx system regulates a broader range of proteins than the GSH system, support distinct function of Trx and GSH in cellular redox control, and show for the first time in mammalian cells selective targeting peptidyl Cys and biological pathways due to deficient function of the Trx system. PMID:23946468

Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation.

PubMed

Metcalfe, Clive; Ramasubramoni, Anjana; Pula, Giordano; Harper, Matthew T; Mundell, Stuart J; Coxon, Carmen H

2016-01-01

Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents.

Probing the Proteome on Earth and Beyond

NASA Astrophysics Data System (ADS)

Ostrom, P.

2008-12-01

Less than a decade ago, protein sequencing was the bane of paleobiology. Since that time researchers have completely sequenced proteins in >50 Ka fossils, been dazzled by reports of collagen peptides in dinosaur bones, and witnessed the development of phylogenetic trees from ancient protein sequences. Enlisting proteomics as biosignature is now in our grasp. In this talk the pitfalls and challenges of mass spectrometric approaches to protein sequencing will be illustrated and phylogenetic applications will be discussed. Work on extinct organisms at Michigan State University, University of Michigan and York University will provide a vantage point to assess methodologies, explore diagenetic alterations, evaluate mass spectra and illustrate issues associated with data base searching. Challenges encountered in the study of paleoproteomics, such as the absence of sequences for extinct organisms in commercially available databases, protein diagenesis and low concentrations of target are parallel to those that will be encountered when protein sequencing is extended to extreme and extraterrestrial environments. Thus, lessons learned from interrogating the ancient proteome are important and necessary step in developing proteomics as a biosignature tools.

Ecological distribution and population physiology defined by proteomics in a natural microbial community

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

Muller, R; Denef, Vincent; Kalnejals, Linda

An important challenge in microbial ecology is developing methods that simultaneously examine the physiology of organisms at the molecular level and their ecosystem level interactions in complex natural systems.We integrated extensive proteomic, geochemical, and biological information from 28 microbial communities collected from an acid mine drainage environment and representing a range of biofilm development stages and geochemical conditions to evaluate how the physiologies of the dominant and less abundant organisms change along environmental gradients. The initial colonist dominates across all environments, but its proteome changes between two stable states as communities diversify, implying that interspecies interactions affect this organism smore » metabolism. Its overall physiology is robust to abiotic environmental factors, but strong correlations exist between these factors and certain subsets of proteins, possibly accounting for its wide environmental distribution. Lower abundance populations are patchier in their distribution, and proteomic data indicate that their environmental niches may be constrained by specific sets of abiotic environmental factors. This research establishes an effective strategy to investigate ecological relationships between microbial physiology and the environment for whole communities in situ« less

Intact mass detection, interpretation, and visualization to automate Top-Down proteomics on a large scale

PubMed Central

Durbin, Kenneth R.; Tran, John C.; Zamdborg, Leonid; Sweet, Steve M. M.; Catherman, Adam D.; Lee, Ji Eun; Li, Mingxi; Kellie, John F.; Kelleher, Neil L.

2011-01-01

Applying high-throughput Top-Down MS to an entire proteome requires a yet-to-be-established model for data processing. Since Top-Down is becoming possible on a large scale, we report our latest software pipeline dedicated to capturing the full value of intact protein data in automated fashion. For intact mass detection, we combine algorithms for processing MS1 data from both isotopically resolved (FT) and charge-state resolved (ion trap) LC-MS data, which are then linked to their fragment ions for database searching using ProSight. Automated determination of human keratin and tubulin isoforms is one result. Optimized for the intricacies of whole proteins, new software modules visualize proteome-scale data based on the LC retention time and intensity of intact masses and enable selective detection of PTMs to automatically screen for acetylation, phosphorylation, and methylation. Software functionality was demonstrated using comparative LC-MS data from yeast strains in addition to human cells undergoing chemical stress. We further these advances as a key aspect of realizing Top-Down MS on a proteomic scale. PMID:20848673

Ecological distribution and population physiology defined by proteomics in a natural microbial community

USGS Publications Warehouse

Mueller, Ryan S.; Denef, Vincent J.; Kalnejais, Linda H.; Suttle, K. Blake; Thomas, Brian C.; Wilmes, Paul; Smith, Richard L.; Nordstrom, D. Kirk; McCleskey, R. Blaine; Shah, Menesh B.; VerBekmoes, Nathan C.; Hettich, Robert L.; Banfield, Jillian F.

2010-01-01

An important challenge in microbial ecology is developing methods that simultaneously examine the physiology of organisms at the molecular level and their ecosystem level interactions in complex natural systems. We integrated extensive proteomic, geochemical, and biological information from 28 microbial communities collected from an acid mine drainage environment and representing a range of biofilm development stages and geochemical conditions to evaluate how the physiologies of the dominant and less abundant organisms change along environmental gradients. The initial colonist dominates across all environments, but its proteome changes between two stable states as communities diversify, implying that interspecies interactions affect this organism's metabolism. Its overall physiology is robust to abiotic environmental factors, but strong correlations exist between these factors and certain subsets of proteins, possibly accounting for its wide environmental distribution. Lower abundance populations are patchier in their distribution, and proteomic data indicate that their environmental niches may be constrained by specific sets of abiotic environmental factors. This research establishes an effective strategy to investigate ecological relationships between microbial physiology and the environment for whole communities in situ.

Bovine Milk Comparative Proteome Analysis from Early, Mid, and Late Lactation in the Cattle Breed, Malnad Gidda (Bos indicus).

PubMed

Mol, Praseeda; Kannegundla, Uday; Dey, Gourav; Gopalakrishnan, Lathika; Dammalli, Manjunath; Kumar, Manish; Patil, Arun H; Basavaraju, Marappa; Rao, Akhila; Ramesha, Kerekoppa P; Prasad, Thottethodi Subrahmanya Keshava

2018-03-01

Bovine milk is important for both veterinary medicine and human nutrition. Understanding the bovine milk proteome at different stages of lactation has therefore broad significance for integrative biology and clinical medicine as well. Indeed, different lactation stages have marked influence on the milk yield, milk constituents, and nourishment of the neonates. We performed a comparative proteome analysis of the bovine milk obtained at different stages of lactation from the Indian indigenous cattle Malnad Gidda (Bos indicus), a widely available breed. The milk differential proteome during the lactation stages in B. indicus has not been investigated to date. Using high-resolution mass spectrometry-based quantitative proteomics of the bovine whey proteins at early, mid, and late lactation stages, we identified a total of 564 proteins, out of which 403 proteins were found to be differentially abundant at different lactation stages. As is expected of any body fluid proteome, 51% of the proteins identified in the milk were found to have signal peptides. Gene ontology analyses were carried out to categorize proteins altered across different lactation stages based on biological process and molecular function, which enabled us to correlate their significance in each lactation stage. We also investigated the potential pathways enriched in different lactation stages using bioinformatics pathway analysis tools. To the best of our knowledge, this study represents the first and largest inventory of milk proteins identified to date for an Indian cattle breed. We believe that the current study broadly informs both veterinary omics research and the emerging field of nutriproteomics during lactation stages.

Comparative proteomics analysis of placenta from pregnant women with intrahepatic cholestasis of pregnancy.

PubMed

Zhang, Ting; Guo, Yueshuai; Guo, Xuejiang; Zhou, Tao; Chen, Daozhen; Xiang, Jingying; Zhou, Zuomin

2013-01-01

Intrahepatic cholestasis of pregnancy (ICP) usually occurs in the third trimester and associated with increased risks in fetal complications. Currently, the exact cause of this disease is unknown. In this study we aim to investigate the potential proteins in placenta, which may participate in the molecular mechanisms of ICP-related fetal complications using iTRAQ-based proteomics approach. The iTRAQ analysis combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to separate differentially expressed placental proteins from 4 pregnant women with ICP and 4 healthy pregnant women. Bioinformatics analysis was used to find the relative processes that these differentially expressed proteins were involved in. Three apoptosis related proteins ERp29, PRDX6 and MPO that resulted from iTRAQ-based proteomics were further verified in placenta by Western blotting and immunohistochemistry. Placental apoptosis was also detected by TUNEL assay. Proteomics results showed there were 38 differentially expressed proteins from pregnant women with ICP and healthy pregnant women, 29 were upregulated and 9 were downregulated in placenta from pregnant women with ICP. Bioinformatics analysis showed most of the identified proteins was functionally related to specific cell processes, including apoptosis, oxidative stress, lipid metabolism. The expression levels of ERp29, PRDX6 and MPO were consistent with the proteomics data. The apoptosis index in placenta from ICP patients was significantly increased. This preliminary work provides a better understanding of the proteomic alterations of placenta from pregnant women with ICP and may provide us some new insights into the pathophysiology and potential novel treatment targets for ICP.

Proteomic Analysis of Serum from Patients with Major Depressive Disorder to Compare Their Depressive and Remission Statuses

PubMed Central

Lee, Jiyeong; Joo, Eun-Jeong; Lim, Hee-Joung; Park, Jong-Moon; Lee, Kyu Young; Park, Arum; Seok, AeEun

2015-01-01

Objective Currently, there are a few biological markers to aid in the diagnosis and treatment of depression. However, it is not sufficient for diagnosis. We attempted to identify differentially expressed proteins during depressive moods as putative diagnostic biomarkers by using quantitative proteomic analysis of serum. Methods Blood samples were collected twice from five patients with major depressive disorder (MDD) at depressive status before treatment and at remission status during treatment. Samples were individually analyzed by liquid chromatography-tandem mass spectrometry for protein profiling. Differentially expressed proteins were analyzed by label-free quantification. Enzyme-linked immunosorbent assay (ELISA) results and receiver-operating characteristic (ROC) curves were used to validate the differentially expressed proteins. For validation, 8 patients with MDD including 3 additional patients and 8 matched normal controls were analyzed. Results The quantitative proteomic studies identified 10 proteins that were consistently upregulated or downregulated in 5 MDD patients. ELISA yielded results consistent with the proteomic analysis for 3 proteins. Expression levels were significantly different between normal controls and MDD patients. The 3 proteins were ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4 and complement component 1qC, which were upregulated during the depressive status. The depressive status could be distinguished from the euthymic status from the ROC curves for these proteins, and this discrimination was enhanced when all 3 proteins were analyzed together. Conclusion This is the first proteomic study in MDD patients to compare intra-individual differences dependent on mood. This technique could be a useful approach to identify MDD biomarkers, but requires additional proteomic studies for validation. PMID:25866527

Translational Research on the Way to Effective Therapy for Alzheimer Disease

PubMed Central

Rosenberg, Roger N.

2006-01-01

Context Alzheimer disease (AD) is a major public health issue with a prediction of 12 million Americans being affected by 2025 from the present 4 million. Molecular and genetic findings have provided significant insights into the roles that amyloid, tau, and apolipoprotein E isoforms have in the causation of AD. A central issue in AD pathogenesis is the amyloid cascade hypothesis. It states that abnormal amyloid processing and accumulation is the primary causative factor of AD and other associated neuropathologic abnormalities are of secondary consequence. It is presented to provide the rationale for novel drug and vaccination therapeutic strategies. Future research directed at prediction and prevention of AD through a genomic and proteomic analysis with identification of multiple polymorphic genes that interact, resulting in increased risk for late-onset AD, are the realistic and ultimate goals. A new approach for drug development is required, one that will emphasize a genomic and proteomic analysis to identify at-risk gene sets whose genetic expression is sufficient to cause late onset, sporadic AD. Prediction and prevention of disease prior to clinical signs and symptoms are the goals. Objective A review and analysis from electronic literature databases and subsequent reference searches of the molecular genetic data including pertinent genetic mutations and abnormal biochemical findings causal of AD, are cited. The amyloid cascade hypothesis, the contributions of apolipoprotein E, and hyperphosphorylated tau are discussed as to their roles in pathogenesis. Molecular targets for potential drug and vaccination therapies are cited from a critical assessment of the molecular and biomedical data. These data form the basis for rational, target-specific drug and vaccination therapies currently employed and planned for the near future. Phase 2 and 3 clinical trial results of drug and vaccination therapies are cited. Conclusions A new approach is needed as current pharmacologic therapy directed at symptomatic relief has proved to be marginally effective. The genomic and proteomic basis of AD will be defined in the near future, and corresponding molecular therapeutic targets will be identified. Genomic neurology has arrived and its application to resolving AD is our best hope. PMID:16275806

Translational research on the way to effective therapy for Alzheimer disease.

PubMed

Rosenberg, Roger N

2005-11-01

Alzheimer disease (AD) is a major public health issue with a prediction of 12 million Americans being affected by 2025 from the present 4 million. Molecular and genetic findings have provided significant insights into the roles that amyloid, tau, and apolipoprotein E isoforms have in the causation of AD. A central issue in AD pathogenesis is the amyloid cascade hypothesis. It states that abnormal amyloid processing and accumulation is the primary causative factor of AD and other associated neuropathologic abnormalities are of secondary consequence. It is presented to provide the rationale for novel drug and vaccination therapeutic strategies. Future research directed at prediction and prevention of AD through a genomic and proteomic analysis with identification of multiple polymorphic genes that interact, resulting in increased risk for late-onset AD, are the realistic and ultimate goals. A new approach for drug development is required, one that will emphasize a genomic and proteomic analysis to identify at-risk gene sets whose genetic expression is sufficient to cause late onset, sporadic AD. Prediction and prevention of disease prior to clinical signs and symptoms are the goals. A review and analysis from electronic literature databases and subsequent reference searches of the molecular genetic data. including pertinent genetic mutations and abnormal biochemical findings causal of AD, are cited. The amyloid cascade hypothesis, the contributions of apolipoprotein E, and hyperphosphorylated tau are discussed as to their roles in pathogenesis. Molecular targets for potential drug and vaccination therapies are cited from a critical assessment of the molecular and biomedical data. These data form the basis for rational, target-specific drug and vaccination therapies currently employed and planned for the near future. Phase 2 and 3 clinical trial results of drug and vaccination therapies are cited. A new approach is needed as current pharmacologic therapy directed at symptomatic relief has proved to be marginally effective. The genomic and proteomic basis of AD will be defined in the near future, and corresponding molecular therapeutic targets will be identified. Genomic neurology has arrived and its application to resolving AD is our best hope.

High-field asymmetric waveform ion mobility spectrometry for mass spectrometry-based proteomics.

PubMed

Swearingen, Kristian E; Moritz, Robert L

2012-10-01

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is an atmospheric pressure ion mobility technique that separates gas-phase ions by their behavior in strong and weak electric fields. FAIMS is easily interfaced with electrospray ionization and has been implemented as an additional separation mode between liquid chromatography (LC) and mass spectrometry (MS) in proteomic studies. FAIMS separation is orthogonal to both LC and MS and is used as a means of on-line fractionation to improve the detection of peptides in complex samples. FAIMS improves dynamic range and concomitantly the detection limits of ions by filtering out chemical noise. FAIMS can also be used to remove interfering ion species and to select peptide charge states optimal for identification by tandem MS. Here, the authors review recent developments in LC-FAIMS-MS and its application to MS-based proteomics.

Application of oncoproteomics to aberrant signalling networks in changing the treatment paradigm in acute lymphoblastic leukaemia.

PubMed

López Villar, Elena; Wang, Xiangdong; Madero, Luis; Cho, William C

2015-01-01

Oncoproteomics is an important innovation in the early diagnosis, management and development of personalized treatment of acute lymphoblastic leukaemia (ALL). As inherent factors are not completely known - e.g. age or family history, radiation exposure, benzene chemical exposure, certain viral exposures such as infection with the human T-cell lymphoma/leukaemia virus-1, as well as some inherited syndromes may raise the risk of ALL - each ALL patient may modify the susceptibility of therapy. Indeed, we consider these unknown inherent factors could be explained via coupling cytogenetics plus proteomics, especially when proteins are the ones which play function within cells. Innovative proteomics to ALL therapy may help to understand the mechanism of drug resistance and toxicities, which in turn will provide some leads to improve ALL management. Most important of these are shotgun proteomic strategies to unravel ALL aberrant signalling networks. Some shotgun proteomic innovations and bioinformatic tools for ALL therapies will be discussed. As network proteins are distinctive characteristics for ALL patients, unrevealed by cytogenetics, those network proteins are currently an important source of novel therapeutic targets that emerge from shotgun proteomics. Indeed, ALL evolution can be studied for each individual patient via oncoproteomics. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Progressing neurobiological strategies against proteostasis failure: Challenges in neurodegeneration.

PubMed

Amanullah, Ayeman; Upadhyay, Arun; Joshi, Vibhuti; Mishra, Ribhav; Jana, Nihar Ranjan; Mishra, Amit

2017-12-01

Proteins are ordered useful cellular entities, required for normal health and organism's survival. The proteome is the absolute set of cellular expressed proteins, which regulates a wide range of physiological functions linked with all domains of life. In aging cells or under unfavorable cellular conditions, misfolding of proteins generates common pathological events linked with neurodegenerative diseases and aging. Current advances of proteome studies systematically generates some progress in our knowledge that how misfolding of proteins or their accumulation can contribute to the impairment or depletion of proteome functions. Still, the underlying causes of this unrecoverable loss are not clear that how such unsolved transitions give rise to multifactorial challengeable degenerative pathological conditions in neurodegeneration. In this review, we specifically focus and systematically summarize various molecular mechanisms of proteostasis maintenance, as well as discuss progressing neurobiological strategies, promising natural and pharmacological candidates, which can be useful to counteract the problem of proteopathies. Our article emphasizes an urgent need that now it is important for us to recognize the fundamentals of proteostasis to design a new molecular framework and fruitful strategies to uncover how the proteome defects are associated with aging and neurodegenerative diseases. A enhance understanding of progress link with proteome and neurobiological challenges may provide new basic concepts in the near future, based on pharmacological agents, linked with impaired proteostasis and neurodegenerative diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Enhanced Detection of Low-Abundance Human Plasma Proteins by Integrating Polyethylene Glycol Fractionation and Immunoaffinity Depletion

PubMed Central

Liu, Haipeng; Yu, Jia; Qiao, Rui; Zhou, Mi; Yang, Yongtao; Zhou, Jian; Xie, Peng

2016-01-01

The enormous depth complexity of the human plasma proteome poses a significant challenge for current mass spectrometry-based proteomic technologies in terms of detecting low-level proteins in plasma, which is essential for successful biomarker discovery efforts. Typically, a single-step analytical approach cannot reduce this intrinsic complexity. Current simplex immunodepletion techniques offer limited capacity for detecting low-abundance proteins, and integrated strategies are thus desirable. In this respect, we developed an improved strategy for analyzing the human plasma proteome by integrating polyethylene glycol (PEG) fractionation with immunoaffinity depletion. PEG fractionation of plasma proteins is simple, rapid, efficient, and compatible with a downstream immunodepletion step. Compared with immunodepletion alone, our integrated strategy substantially improved the proteome coverage afforded by PEG fractionation. Coupling this new protocol with liquid chromatography-tandem mass spectrometry, 135 proteins with reported normal concentrations below 100 ng/mL were confidently identified as common low-abundance proteins. A side-by-side comparison indicated that our integrated strategy was increased by average 43.0% in the identification rate of low-abundance proteins, relying on an average 65.8% increase of the corresponding unique peptides. Further investigation demonstrated that this combined strategy could effectively alleviate the signal-suppressive effects of the major high-abundance proteins by affinity depletion, especially with moderate-abundance proteins after incorporating PEG fractionation, thereby greatly enhancing the detection of low-abundance proteins. In sum, the newly developed strategy of incorporating PEG fractionation to immunodepletion methods can potentially aid in the discovery of plasma biomarkers of therapeutic and clinical interest. PMID:27832179

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

Environmental Interactions and Epistasis Are Revealed in the Proteomic Responses to Complex Stimuli

PubMed Central

Samir, Parimal; Rahul; Slaughter, James C.; Link, Andrew J.

2015-01-01

Ultimately, the genotype of a cell and its interaction with the environment determine the cell’s biochemical state. While the cell’s response to a single stimulus has been studied extensively, a conceptual framework to model the effect of multiple environmental stimuli applied concurrently is not as well developed. In this study, we developed the concepts of environmental interactions and epistasis to explain the responses of the S. cerevisiae proteome to simultaneous environmental stimuli. We hypothesize that, as an abstraction, environmental stimuli can be treated as analogous to genetic elements. This would allow modeling of the effects of multiple stimuli using the concepts and tools developed for studying gene interactions. Mirroring gene interactions, our results show that environmental interactions play a critical role in determining the state of the proteome. We show that individual and complex environmental stimuli behave similarly to genetic elements in regulating the cellular responses to stimuli, including the phenomena of dominance and suppression. Interestingly, we observed that the effect of a stimulus on a protein is dominant over other stimuli if the response to the stimulus involves the protein. Using publicly available transcriptomic data, we find that environmental interactions and epistasis regulate transcriptomic responses as well. PMID:26247773

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

Quantitative proteomics and dynamic imaging of the nucleolus reveal distinct responses to UV and ionizing radiation.

PubMed

Moore, Henna M; Bai, Baoyan; Boisvert, François-Michel; Latonen, Leena; Rantanen, Ville; Simpson, Jeremy C; Pepperkok, Rainer; Lamond, Angus I; Laiho, Marikki

2011-10-01

The nucleolus is a nuclear organelle that coordinates rRNA transcription and ribosome subunit biogenesis. Recent proteomic analyses have shown that the nucleolus contains proteins involved in cell cycle control, DNA processing and DNA damage response and repair, in addition to the many proteins connected with ribosome subunit production. Here we study the dynamics of nucleolar protein responses in cells exposed to stress and DNA damage caused by ionizing and ultraviolet (UV) radiation in diploid human fibroblasts. We show using a combination of imaging and quantitative proteomics methods that nucleolar substructure and the nucleolar proteome undergo selective reorganization in response to UV damage. The proteomic responses to UV include alterations of functional protein complexes such as the SSU processome and exosome, and paraspeckle proteins, involving both decreases and increases in steady state protein ratios, respectively. Several nonhomologous end-joining proteins (NHEJ), such as Ku70/80, display similar fast responses to UV. In contrast, nucleolar proteomic responses to IR are both temporally and spatially distinct from those caused by UV, and more limited in terms of magnitude. With the exception of the NHEJ and paraspeckle proteins, where IR induces rapid and transient changes within 15 min of the damage, IR does not alter the ratios of most other functional nucleolar protein complexes. The rapid transient decrease of NHEJ proteins in the nucleolus indicates that it may reflect a response to DNA damage. Our results underline that the nucleolus is a specific stress response organelle that responds to different damage and stress agents in a unique, damage-specific manner.

Proteomic analysis of human aqueous humor using multidimensional protein identification technology

PubMed Central

Richardson, Matthew R.; Price, Marianne O.; Price, Francis W.; Pardo, Jennifer C.; Grandin, Juan C.; You, Jinsam; Wang, Mu

2009-01-01

Aqueous humor (AH) supports avascular tissues in the anterior segment of the eye, maintains intraocular pressure, and potentially influences the pathogenesis of ocular diseases. Nevertheless, the AH proteome is still poorly defined despite several previous efforts, which were hindered by interfering high abundance proteins, inadequate animal models, and limited proteomic technologies. To facilitate future investigations into AH function, the AH proteome was extensively characterized using an advanced proteomic approach. Samples from patients undergoing cataract surgery were pooled and depleted of interfering abundant proteins and thereby divided into two fractions: albumin-bound and albumin-depleted. Multidimensional Protein Identification Technology (MudPIT) was utilized for each fraction; this incorporates strong cation exchange chromatography to reduce sample complexity before reversed-phase liquid chromatography and tandem mass spectrometric analysis. Twelve proteins had multi-peptide, high confidence identifications in the albumin-bound fraction and 50 proteins had multi-peptide, high confidence identifications in the albumin-depleted fraction. Gene ontological analyses were performed to determine which cellular components and functions were enriched. Many proteins were previously identified in the AH and for several their potential role in the AH has been investigated; however, the majority of identified proteins were novel and only speculative roles can be suggested. The AH was abundant in anti-oxidant and immunoregulatory proteins as well as anti-angiogenic proteins, which may be involved in maintaining the avascular tissues. This is the first known report to extensively characterize and describe the human AH proteome and lays the foundation for future work regarding its function in homeostatic and pathologic states. PMID:20019884

Review of software tools for design and analysis of large scale MRM proteomic datasets.

PubMed

Colangelo, Christopher M; Chung, Lisa; Bruce, Can; Cheung, Kei-Hoi

2013-06-15

Selective or Multiple Reaction monitoring (SRM/MRM) is a liquid-chromatography (LC)/tandem-mass spectrometry (MS/MS) method that enables the quantitation of specific proteins in a sample by analyzing precursor ions and the fragment ions of their selected tryptic peptides. Instrumentation software has advanced to the point that thousands of transitions (pairs of primary and secondary m/z values) can be measured in a triple quadrupole instrument coupled to an LC, by a well-designed scheduling and selection of m/z windows. The design of a good MRM assay relies on the availability of peptide spectra from previous discovery-phase LC-MS/MS studies. The tedious aspect of manually developing and processing MRM assays involving thousands of transitions has spurred to development of software tools to automate this process. Software packages have been developed for project management, assay development, assay validation, data export, peak integration, quality assessment, and biostatistical analysis. No single tool provides a complete end-to-end solution, thus this article reviews the current state and discusses future directions of these software tools in order to enable researchers to combine these tools for a comprehensive targeted proteomics workflow. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Biomarkers in prostate cancer: what’s new?

PubMed Central

Sartori, David A.; Chan, Daniel W.

2014-01-01

Purpose of review This review is intended to provide an overview of the current state of biomarkers for prostate cancer (PCa), with a focus on biomarkers approved by the US Food and Drug Administration (FDA) as well as biomarkers available from Clinical Laboratory Improvement Amendment (CLIA)-certified clinical laboratories within the last 1–2 years. Recent findings During the past 2 years, two biomarkers have been approved by the US FDA. These include proPSA as part of the Prostate Health Index (phi) by Beckman Coulter, Inc and PCA3 as Progensa by Gen Probe, Inc. With the advances in genomic and proteomic technologies, several new CLIA-based laboratory-developed tests have become available. Examples are Oncotype DX from Genomics Health, Inc, and Prolaris from Myriad Genetics, Inc. In most cases, these new tests are based on a combination of multiple genomic or proteomic biomarkers. Summary Several new tests, as discussed in this review, have become available during the last 2 years. Although the intended use of most of these tests is to distinguish PCa from benign prostatic conditions with better sensitivity and specificity than prostate-specific antigen, studies have shown that some of them may also be useful in the differentiation of aggressive from nonaggressive forms of PCa. PMID:24626128

Recent Achievements in Characterizing the Histone Code and Approaches to Integrating Epigenomics and Systems Biology.

PubMed

Janssen, K A; Sidoli, S; Garcia, B A

2017-01-01

Functional epigenetic regulation occurs by dynamic modification of chromatin, including genetic material (i.e., DNA methylation), histone proteins, and other nuclear proteins. Due to the highly complex nature of the histone code, mass spectrometry (MS) has become the leading technique in identification of single and combinatorial histone modifications. MS has now overcome antibody-based strategies due to its automation, high resolution, and accurate quantitation. Moreover, multiple approaches to analysis have been developed for global quantitation of posttranslational modifications (PTMs), including large-scale characterization of modification coexistence (middle-down and top-down proteomics), which is not currently possible with any other biochemical strategy. Recently, our group and others have simplified and increased the effectiveness of analyzing histone PTMs by improving multiple MS methods and data analysis tools. This review provides an overview of the major achievements in the analysis of histone PTMs using MS with a focus on the most recent improvements. We speculate that the workflow for histone analysis at its state of the art is highly reliable in terms of identification and quantitation accuracy, and it has the potential to become a routine method for systems biology thanks to the possibility of integrating histone MS results with genomics and proteomics datasets. © 2017 Elsevier Inc. All rights reserved.

Seminal plasma proteomes and sperm fertility.

PubMed

Druart, Xavier; de Graaf, Simon

2018-04-10

During ejaculation, the spermatozoa are transported by the seminal plasma, a fluid resulting from secretions originating mainly from the prostate and the seminal vesicles in mammals. The interaction of the seminal plasma with spermatozoa induces binding of seminal proteins onto the sperm surface and membrane remodeling potentially impacting the sperm transport, survival and fertilizing ability in the female genital tract. The seminal plasma also contains peptides and proteins involved in the inflammatory and immune response of the female tract. Therefore the seminal plasma proteome has been investigated in a large range of taxa, including mammals, birds, fishes and insect species. The association of the seminal plasma with semen preservation or fertility identified proteic markers of seminal plasma function in domestic species. This review summarizes the current knowledge in seminal plasma proteomes and proteic markers of sperm preservation in animal species. Copyright © 2018. Published by Elsevier B.V.

Cardioproteomics: advancing the discovery of signaling mechanisms involved in cardiovascular diseases

PubMed Central

Cui, Ziyou; Dewey, Shannamar; Gomes, Aldrin V

2011-01-01

Cardioproteomics (Cardiovascular proteomics) is fast becoming an indispensible technique in deciphering changes in signaling pathways that occur in cardiovascular diseases (CVDs). The quality and availability of the instruments and bioinformatics software used for cardioproteomics continues to improve, and these techniques are now available to most cardiovascular researchers either directly or indirectly via university core centers. The heart and aorta are specialized tissues which present unique challenges to investigate. Currently, the diverse range of proteomic techniques available for cardiovascular research makes the choice of the best method or best combination of methods for the disease parameter(s) being investigated as important as the equipment used. This review focuses on proteomic techniques and their applications which have advanced our understanding of the signaling mechanisms involved in CVDs at the levels of protein complex/protein-protein interaction, post-translational modifications and signaling induced protein changes. PMID:22254205

Candidate-based proteomics in the search for biomarkers of cardiovascular disease

PubMed Central

Anderson, Leigh

2005-01-01

The key concept of proteomics (looking at many proteins at once) opens new avenues in the search for clinically useful biomarkers of disease, treatment response and ageing. As the number of proteins that can be detected in plasma or serum (the primary clinical diagnostic samples) increases towards 1000, a paradoxical decline has occurred in the number of new protein markers approved for diagnostic use in clinical laboratories. This review explores the limitations of current proteomics protein discovery platforms, and proposes an alternative approach, applicable to a range of biological/physiological problems, in which quantitative mass spectrometric methods developed for analytical chemistry are employed to measure limited sets of candidate markers in large sets of clinical samples. A set of 177 candidate biomarker proteins with reported associations to cardiovascular disease and stroke are presented as a starting point for such a ‘directed proteomics’ approach. PMID:15611012

Evaluation of several two-dimensional gel electrophoresis techniques in cardiac proteomics.

PubMed

Li, Zhao Bo; Flint, Paul W; Boluyt, Marvin O

2005-09-01

Two-dimensional gel electrophoresis (2-DE) is currently the best method for separating complex mixtures of proteins, and its use is gradually becoming more common in cardiac proteome analysis. A number of variations in basic 2-DE have emerged, but their usefulness in analyzing cardiac tissue has not been evaluated. The purpose of the present study was to systematically evaluate the capabilities and limitations of several 2-DE techniques for separating proteins from rat heart tissue. Immobilized pH gradient strips of various pH ranges, parameters of protein loading and staining, subcellular fractionation, and detection of phosphorylated proteins were studied. The results provide guidance for proteome analysis of cardiac and other tissues in terms of selection of the isoelectric point separating window for cardiac proteins, accurate quantitation of cardiac protein abundance, stabilization of technical variation, reduction of sample complexity, enrichment of low-abundant proteins, and detection of phosphorylated proteins.

Proteomics and metabolomics for mechanistic insights and biomarker discovery in cardiovascular disease.

PubMed

Barallobre-Barreiro, Javier; Chung, Yuen-Li; Mayr, Manuel

2013-08-01

In the last decade, proteomics and metabolomics have contributed substantially to our understanding of cardiovascular diseases. The unbiased assessment of pathophysiological processes without a priori assumptions complements other molecular biology techniques that are currently used in a reductionist approach. In this review, we highlight some of the "omics" methods used to assess protein and metabolite changes in cardiovascular disease. A discrete biological function is very rarely attributed to a single molecule; more often it is the combined input of many proteins. In contrast to the reductionist approach, in which molecules are studied individually, "omics" platforms allow the study of more complex interactions in biological systems. Combining proteomics and metabolomics to quantify changes in metabolites and their corresponding enzymes will advance our understanding of pathophysiological mechanisms and aid the identification of novel biomarkers for cardiovascular disease. Copyright © 2013 Sociedad Española de Cardiología. Published by Elsevier Espana. All rights reserved.

Approaches for Defining the Hsp90-dependent Proteome

PubMed Central

Hartson, Steven D.; Matts, Robert L.

2011-01-01

Hsp90 is the target of ongoing drug discovery studies seeking new compounds to treat cancer, neurodegenerative diseases, and protein folding disorders. To better understand Hsp90’s roles in cellular pathologies and in normal cells, numerous studies have utilized proteomics assays and related high-throughput tools to characterize its physical and functional protein partnerships. This review surveys these studies, and summarizes the strengths and limitations of the individual attacks. We also include downloadable spreadsheets compiling all of the Hsp90-interacting proteins identified in more than 23 studies. These tools include cross-references among gene aliases, human homologues of yeast Hsp90-interacting proteins, hyperlinks to database entries, summaries of canonical pathways that are enriched in the Hsp90 interactome, and additional bioinformatic annotations. In addition to summarizing Hsp90 proteomics studies performed to date and the insights they have provided, we identify gaps in our current understanding of Hsp90-mediated proteostasis. PMID:21906632

Methods, Tools and Current Perspectives in Proteogenomics *

PubMed Central

Ruggles, Kelly V.; Krug, Karsten; Wang, Xiaojing; Clauser, Karl R.; Wang, Jing; Payne, Samuel H.; Fenyö, David; Zhang, Bing; Mani, D. R.

2017-01-01

With combined technological advancements in high-throughput next-generation sequencing and deep mass spectrometry-based proteomics, proteogenomics, i.e. the integrative analysis of proteomic and genomic data, has emerged as a new research field. Early efforts in the field were focused on improving protein identification using sample-specific genomic and transcriptomic sequencing data. More recently, integrative analysis of quantitative measurements from genomic and proteomic studies have identified novel insights into gene expression regulation, cell signaling, and disease. Many methods and tools have been developed or adapted to enable an array of integrative proteogenomic approaches and in this article, we systematically classify published methods and tools into four major categories, (1) Sequence-centric proteogenomics; (2) Analysis of proteogenomic relationships; (3) Integrative modeling of proteogenomic data; and (4) Data sharing and visualization. We provide a comprehensive review of methods and available tools in each category and highlight their typical applications. PMID:28456751

Clinical potential of proteomics in the diagnosis of ovarian cancer.

PubMed

Ardekani, Ali M; Liotta, Lance A; Petricoin, Emanuel F

2002-07-01

The need for specific and sensitive markers of ovarian cancer is critical. Finding a sensitive and specific test for its detection has an important public health impact. Currently, there are no effective screening options available for patients with ovarian cancer. CA-125, the most widely used biomarker for ovarian cancer, does not have a high positive predictive value and it is only effective when used in combination with other diagnostic tests. However, pathologic changes taking place within the ovary may be reflected in biomarker patterns in the serum. Combination of mass spectra generated by new proteomic technologies, such as surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF) and artificial-intelligence-based informatic algorithms, have been used to discover a small set of key protein values and discriminate normal from ovarian cancer patients. Serum proteomic pattern analysis might be applied ultimately in medical screening clinics, as a supplement to the diagnostic work-up and evaluation.

Current Challenges in Detecting Food Allergens by Shotgun and Targeted Proteomic Approaches: A Case Study on Traces of Peanut Allergens in Baked Cookies

PubMed Central

Pedreschi, Romina; Nørgaard, Jørgen; Maquet, Alain

2012-01-01

There is a need for selective and sensitive methods to detect the presence of food allergens at trace levels in highly processed food products. In this work, a combination of non-targeted and targeted proteomics approaches are used to illustrate the difficulties encountered in the detection of the major peanut allergens Ara h 1, Ara h 2 and Ara h 3 from a representative processed food matrix. Shotgun proteomics was employed for selection of the proteotypic peptides for targeted approaches via selective reaction monitoring. Peanut presence through detection of the proteotypic Ara h 3/4 peptides AHVQVVDSNGNR (m/z 432.5, 3+) and SPDIYNPQAGSLK (m/z 695.4, 2+) was confirmed and the developed method was able to detect peanut presence at trace levels (≥10 μg peanut g−1 matrix) in baked cookies. PMID:22413066

Proteomic Definitions of Mesenchymal Stem Cells

PubMed Central

Maurer, Martin H.

2011-01-01

Mesenchymal stem cells (MSCs) are pluripotent cells isolated from the bone marrow and various other organs. They are able to proliferate and self-renew, as well as to give rise to progeny of at least the osteogenic, chondrogenic, and adipogenic lineages. Despite this functional definition, MSCs can also be defined by their expression of a distinct set of cell surface markers. In the current paper, studies investigating the proteome of human MSCs are reviewed with the aim to identify common protein markers of MSCs. The proteomic analysis of MSCs revealed a distinct set of proteins representing the basic molecular inventory, including proteins for (i) cell surface markers, (ii) the responsiveness to growth factors, (iii) the reuse of developmental signaling cascades in adult stem cells, (iv) the interaction with molecules of the extracellular matrix, (v) the expression of genes regulating transcription and translation, (vi) the control of the cell number, and (vii) the protection against cellular stress. PMID:21437194

Proteomics links the redox state to calcium signaling during bleaching of the scleractinian coral Acropora microphthalma on exposure to high solar irradiance and thermal stress.

PubMed

Weston, Andrew J; Dunlap, Walter C; Beltran, Victor H; Starcevic, Antonio; Hranueli, Daslav; Ward, Malcolm; Long, Paul F

2015-03-01

Shipboard experiments were each performed over a 2 day period to examine the proteomic response of the symbiotic coral Acropora microphthalma exposed to acute conditions of high temperature/low light or high light/low temperature stress. During these treatments, corals had noticeably bleached. The photosynthetic performance of residual algal endosymbionts was severely impaired but showed signs of recovery in both treatments by the end of the second day. Changes in the coral proteome were determined daily and, using recently available annotated genome sequences, the individual contributions of the coral host and algal endosymbionts could be extracted from these data. Quantitative changes in proteins relevant to redox state and calcium metabolism are presented. Notably, expression of common antioxidant proteins was not detected from the coral host but present in the algal endosymbiont proteome. Possible roles for elevated carbonic anhydrase in the coral host are considered: to restore intracellular pH diminished by loss of photosynthetic activity, to indirectly limit intracellular calcium influx linked with enhanced calmodulin expression to impede late-stage symbiont exocytosis, or to enhance inorganic carbon transport to improve the photosynthetic performance of algal symbionts that remain in hospite. Protein effectors of calcium-dependent exocytosis were present in both symbiotic partners. No caspase-family proteins associated with host cell apoptosis, with exception of the autophagy chaperone HSP70, were detected, suggesting that algal loss and photosynthetic dysfunction under these experimental conditions were not due to host-mediated phytosymbiont destruction. Instead, bleaching occurred by symbiont exocytosis and loss of light-harvesting pigments of algae that remain in hospite. These proteomic data are, therefore, consistent with our premise that coral endosymbionts can mediate their own retention or departure from the coral host, which may manifest as "symbiont shuffling" of Symbiodinium clades in response to environmental stress. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Colonization State Influences the Hemocyte Proteome in a Beneficial Squid–Vibrio Symbiosis*

PubMed Central

Schleicher, Tyler R.; VerBerkmoes, Nathan C.; Shah, Manesh; Nyholm, Spencer V.

2014-01-01

The squid Euprymna scolopes and the luminescent bacterium Vibrio fischeri form a highly specific beneficial light organ symbiosis. Not only does the host have to select V. fischeri from the environment, but it must also prevent subsequent colonization by non-symbiotic microorganisms. Host macrophage-like hemocytes are believed to play a role in mediating the symbiosis with V. fischeri. Previous studies have shown that the colonization state of the light organ influences the host's hemocyte response to the symbiont. To further understand the molecular mechanisms behind this process, we used two quantitative mass-spectrometry-based proteomic techniques, isobaric tags for relative and absolute quantification (iTRAQ) and label-free spectral counting, to compare and quantify the adult hemocyte proteomes from colonized (sym) and uncolonized (antibiotic-treated/cured) squid. Overall, iTRAQ allowed for the quantification of 1,024 proteins with two or more peptides. Thirty-seven unique proteins were determined to be significantly different between sym and cured hemocytes (p value < 0.05), with 20 more abundant proteins and 17 less abundant in sym hemocytes. The label-free approach resulted in 1,241 proteins that were identified in all replicates. Of 185 unique proteins present at significantly different amounts in sym hemocytes (as determined by spectral counting), 92 were more abundant and 93 were less abundant. Comparisons between iTRAQ and spectral counting revealed that 30 of the 37 proteins quantified via iTRAQ exhibited trends similar to those identified by the label-free method. Both proteomic techniques mutually identified 16 proteins that were significantly different between the two groups of hemocytes (p value < 0.05). The presence of V. fischeri in the host light organ influenced the abundance of proteins associated with the cytoskeleton, adhesion, lysosomes, proteolysis, and the innate immune response. These data provide evidence that colonization by V. fischeri alters the hemocyte proteome and reveals proteins that may be important for maintaining host–symbiont specificity. PMID:25038065

Proteomic analysis of tardigrades: towards a better understanding of molecular mechanisms by anhydrobiotic organisms.

PubMed

Schokraie, Elham; Hotz-Wagenblatt, Agnes; Warnken, Uwe; Mali, Brahim; Frohme, Marcus; Förster, Frank; Dandekar, Thomas; Hengherr, Steffen; Schill, Ralph O; Schnölzer, Martina

2010-03-03

Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions. The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades.

Proteomic Analysis of Tardigrades: Towards a Better Understanding of Molecular Mechanisms by Anhydrobiotic Organisms

PubMed Central

Schokraie, Elham; Hotz-Wagenblatt, Agnes; Warnken, Uwe; Mali, Brahim; Frohme, Marcus; Förster, Frank; Dandekar, Thomas; Hengherr, Steffen; Schill, Ralph O.; Schnölzer, Martina

2010-01-01

Background Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. Principal Findings Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions. Conclusions The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades. PMID:20224743

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.

The Hemolymph Proteome of Fed and Starved Drosophila Larvae

PubMed Central

Goetze, Sandra; Ahrens, Christian H.; Omasits, Ulrich; Marty, Florian; Simigdala, Nikiana; Meyer, Imke; Wollscheid, Bernd; Brunner, Erich; Hafen, Ernst; Lehner, Christian F.

2013-01-01

The co-operation of specialized organ systems in complex multicellular organisms depends on effective chemical communication. Thus, body fluids (like blood, lymph or intraspinal fluid) contain myriads of signaling mediators apart from metabolites. Moreover, these fluids are also of crucial importance for immune and wound responses. Compositional analyses of human body fluids are therefore of paramount diagnostic importance. Further improving their comprehensiveness should increase our understanding of inter-organ communication. In arthropods, which have trachea for gas exchange and an open circulatory system, the single dominating interstitial fluid is the hemolymph. Accordingly, a detailed analysis of hemolymph composition should provide an especially comprehensive picture of chemical communication and defense in animals. Therefore we used an extensive protein fractionation workflow in combination with a discovery-driven proteomic approach to map out the detectable protein composition of hemolymph isolated from Drosophila larvae. Combined mass spectrometric analysis revealed more than 700 proteins extending far beyond the previously known Drosophila hemolymph proteome. Moreover, by comparing hemolymph isolated from either fed or starved larvae, we provide initial provisional insights concerning compositional changes in response to nutritional state. Storage proteins in particular were observed to be strongly reduced by starvation. Our hemolymph proteome catalog provides a rich basis for data mining, as exemplified by our identification of potential novel cytokines, as well as for future quantitative analyses by targeted proteomics. PMID:23840627

Less is More: Membrane Protein Digestion Beyond Urea–Trypsin Solution for Next-level Proteomics*

PubMed Central

Zhang, Xi

2015-01-01

The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea–trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting—not destroying—structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. PMID:26081834

The hemolymph proteome of fed and starved Drosophila larvae.

PubMed

Handke, Björn; Poernbacher, Ingrid; Goetze, Sandra; Ahrens, Christian H; Omasits, Ulrich; Marty, Florian; Simigdala, Nikiana; Meyer, Imke; Wollscheid, Bernd; Brunner, Erich; Hafen, Ernst; Lehner, Christian F

2013-01-01

The co-operation of specialized organ systems in complex multicellular organisms depends on effective chemical communication. Thus, body fluids (like blood, lymph or intraspinal fluid) contain myriads of signaling mediators apart from metabolites. Moreover, these fluids are also of crucial importance for immune and wound responses. Compositional analyses of human body fluids are therefore of paramount diagnostic importance. Further improving their comprehensiveness should increase our understanding of inter-organ communication. In arthropods, which have trachea for gas exchange and an open circulatory system, the single dominating interstitial fluid is the hemolymph. Accordingly, a detailed analysis of hemolymph composition should provide an especially comprehensive picture of chemical communication and defense in animals. Therefore we used an extensive protein fractionation workflow in combination with a discovery-driven proteomic approach to map out the detectable protein composition of hemolymph isolated from Drosophila larvae. Combined mass spectrometric analysis revealed more than 700 proteins extending far beyond the previously known Drosophila hemolymph proteome. Moreover, by comparing hemolymph isolated from either fed or starved larvae, we provide initial provisional insights concerning compositional changes in response to nutritional state. Storage proteins in particular were observed to be strongly reduced by starvation. Our hemolymph proteome catalog provides a rich basis for data mining, as exemplified by our identification of potential novel cytokines, as well as for future quantitative analyses by targeted proteomics.

LFQProfiler and RNP(xl): Open-Source Tools for Label-Free Quantification and Protein-RNA Cross-Linking Integrated into Proteome Discoverer.

PubMed

Veit, Johannes; Sachsenberg, Timo; Chernev, Aleksandar; Aicheler, Fabian; Urlaub, Henning; Kohlbacher, Oliver

2016-09-02

Modern mass spectrometry setups used in today's proteomics studies generate vast amounts of raw data, calling for highly efficient data processing and analysis tools. Software for analyzing these data is either monolithic (easy to use, but sometimes too rigid) or workflow-driven (easy to customize, but sometimes complex). Thermo Proteome Discoverer (PD) is a powerful software for workflow-driven data analysis in proteomics which, in our eyes, achieves a good trade-off between flexibility and usability. Here, we present two open-source plugins for PD providing additional functionality: LFQProfiler for label-free quantification of peptides and proteins, and RNP(xl) for UV-induced peptide-RNA cross-linking data analysis. LFQProfiler interacts with existing PD nodes for peptide identification and validation and takes care of the entire quantitative part of the workflow. We show that it performs at least on par with other state-of-the-art software solutions for label-free quantification in a recently published benchmark ( Ramus, C.; J. Proteomics 2016 , 132 , 51 - 62 ). The second workflow, RNP(xl), represents the first software solution to date for identification of peptide-RNA cross-links including automatic localization of the cross-links at amino acid resolution and localization scoring. It comes with a customized integrated cross-link fragment spectrum viewer for convenient manual inspection and validation of the results.

Using Public Data for Comparative Proteome Analysis in Precision Medicine Programs.

PubMed

Hughes, Christopher S; Morin, Gregg B

2018-03-01

Maximizing the clinical utility of information obtained in longitudinal precision medicine programs would benefit from robust comparative analyses to known information to assess biological features of patient material toward identifying the underlying features driving their disease phenotype. Herein, the potential for utilizing publically deposited mass-spectrometry-based proteomics data to perform inter-study comparisons of cell-line or tumor-tissue materials is investigated. To investigate the robustness of comparison between MS-based proteomics studies carried out with different methodologies, deposited data representative of label-free (MS1) and isobaric tagging (MS2 and MS3 quantification) are utilized. In-depth quantitative proteomics data acquired from analysis of ovarian cancer cell lines revealed the robust recapitulation of observable gene expression dynamics between individual studies carried out using significantly different methodologies. The observed signatures enable robust inter-study clustering of cell line samples. In addition, the ability to classify and cluster tumor samples based on observed gene expression trends when using a single patient sample is established. With this analysis, relevant gene expression dynamics are obtained from a single patient tumor, in the context of a precision medicine analysis, by leveraging a large cohort of repository data as a comparator. Together, these data establish the potential for state-of-the-art MS-based proteomics data to serve as resources for robust comparative analyses in precision medicine applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A proteome-scale map of the human interactome network

PubMed Central

Rolland, Thomas; Taşan, Murat; Charloteaux, Benoit; Pevzner, Samuel J.; Zhong, Quan; Sahni, Nidhi; Yi, Song; Lemmens, Irma; Fontanillo, Celia; Mosca, Roberto; Kamburov, Atanas; Ghiassian, Susan D.; Yang, Xinping; Ghamsari, Lila; Balcha, Dawit; Begg, Bridget E.; Braun, Pascal; Brehme, Marc; Broly, Martin P.; Carvunis, Anne-Ruxandra; Convery-Zupan, Dan; Corominas, Roser; Coulombe-Huntington, Jasmin; Dann, Elizabeth; Dreze, Matija; Dricot, Amélie; Fan, Changyu; Franzosa, Eric; Gebreab, Fana; Gutierrez, Bryan J.; Hardy, Madeleine F.; Jin, Mike; Kang, Shuli; Kiros, Ruth; Lin, Guan Ning; Luck, Katja; MacWilliams, Andrew; Menche, Jörg; Murray, Ryan R.; Palagi, Alexandre; Poulin, Matthew M.; Rambout, Xavier; Rasla, John; Reichert, Patrick; Romero, Viviana; Ruyssinck, Elien; Sahalie, Julie M.; Scholz, Annemarie; Shah, Akash A.; Sharma, Amitabh; Shen, Yun; Spirohn, Kerstin; Tam, Stanley; Tejeda, Alexander O.; Trigg, Shelly A.; Twizere, Jean-Claude; Vega, Kerwin; Walsh, Jennifer; Cusick, Michael E.; Xia, Yu; Barabási, Albert-László; Iakoucheva, Lilia M.; Aloy, Patrick; De Las Rivas, Javier; Tavernier, Jan; Calderwood, Michael A.; Hill, David E.; Hao, Tong; Roth, Frederick P.; Vidal, Marc

2014-01-01

SUMMARY Just as reference genome sequences revolutionized human genetics, reference maps of interactome networks will be critical to fully understand genotype-phenotype relationships. Here, we describe a systematic map of ~14,000 high-quality human binary protein-protein interactions. At equal quality, this map is ~30% larger than what is available from small-scale studies published in the literature in the last few decades. While currently available information is highly biased and only covers a relatively small portion of the proteome, our systematic map appears strikingly more homogeneous, revealing a “broader” human interactome network than currently appreciated. The map also uncovers significant inter-connectivity between known and candidate cancer gene products, providing unbiased evidence for an expanded functional cancer landscape, while demonstrating how high quality interactome models will help “connect the dots” of the genomic revolution. PMID:25416956

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.

Nutritional mitigation of winter thermal stress in gilthead seabream: Associated metabolic pathways and potential indicators of nutritional state.

PubMed

Richard, Nadège; Silva, Tomé S; Wulff, Tune; Schrama, Denise; Dias, Jorge P; Rodrigues, Pedro M L; Conceição, Luís E C

2016-06-16

A trial was carried out with gilthead seabream juveniles, aiming to investigate the ability of an enhanced dietary formulation (diet Winter Feed, WF, containing a higher proportion of marine-derived protein sources and supplemented in phospholipids, vitamin C, vitamin E and taurine) to assist fish in coping with winter thermal stress, compared to a low-cost commercial diet (diet CTRL). In order to identify the metabolic pathways affected by WF diet, a comparative two dimensional differential in-gel electrophoresis (2D-DIGE) analysis of fish liver proteome (pH 4–7) was undertaken at the end of winter. A total of 404 protein spots, out of 1637 detected, were differentially expressed between the two groups of fish. Mass spectrometry analysis of selected spots suggested that WF diet improved oxidative stress defense, reduced endoplasmic reticulum stress, enhanced metabolic flux through methionine cycle and phenylalanine/tyrosine catabolism, and induced higher aerobic metabolism and gluconeogenesis. Results support the notion that WF diet had a positive effect on fish nutritional state by partially counteracting the effect of thermal stress and underlined the sensitivity of proteome data for nutritional and metabolic profiling purposes. Intragroup variability and co-measured information were also used to pinpoint which proteins displayed a stronger relation with fish nutritional state. Winter low water temperature is a critical factor for gilthead seabream farming in the Mediterranean region, leading to a reduction of feed intake, which often results in metabolic and immunological disorders and stagnation of growth performances. In a recent trial, we investigated the ability of an enhanced dietary formulation (diet WF) to assist gilthead seabream in coping with winter thermal stress, compared to a standard commercial diet (diet CTRL). Within this context, in the present work, we identified metabolic processes that are involved in the stress-mitigating effect observed with diet WF, by undertaking a comparative analysis of fish liver proteome at the end of winter. This study brings information relative to biological processes that are involved in gilthead seabream winter thermal stress and shows that these can be mitigated through a nutritional strategy, assisting gilthead seabream to deal better with winter thermal conditions. Furthermore, the results show that proteomic information not only clearly distinguishes the two dietary groups from each other, but also captures heterogeneities that reflect intra-group differences in nutritional state. This was exploited in this work to refine the variable selection strategy so that protein spots displaying a stronger correlation with “nutritional state” could be identified as possible indicators of gilthead seabream metabolic and nutritional state. Finally, this study shows that gel-based proteomics seems to provide more reliable information than transmissive FT-IR spectroscopy, for the purposes of nutritional and metabolic profiling.

Combining Capillary Electrophoresis with Mass Spectrometry for Applications in Proteomics

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

Simpson, David C.; Smith, Richard D.

2005-04-01

Throughout the field of global proteomics, ranging from simple organism studies to human medical applications, the high sample complexity creates demands for improved separations and analysis techniques. Furthermore, with increased organism complexity, the correlation between proteome and genome becomes less certain due to extensive mRNA processing prior to translation. In this way, the same DNA sequence can potentially code for regions in a number of distinct proteins; quantitative differences in expression (or abundance) between these often-related species are of significant interest. Well-established proteomics techniques, which use genomic information to identify peptides that originate from protease digestion, often cannot easily distinguishmore » between such gene products; intact protein-level analyses are required to complete the picture, particularly for identifying post-translational modifications. While chromatographic techniques are currently better suited to peptide analysis, capillary electrophoresis (CE) in combination with mass spectrometry (MS) may become important for intact protein analysis. This review focuses on CE/MS instrumentation and techniques showing promise for such applications, highlighting those with greatest potential. Reference will also be made to developments relevant to peptide-level analyses for use in time- or sample-limited situations.« less

Proteomic analysis of post translational modifications in cyanobacteria.

PubMed

Xiong, Qian; Chen, Zhuo; Ge, Feng

2016-02-16

Cyanobacteria are a diverse group of Gram-negative bacteria and the only prokaryotes capable of oxygenic photosynthesis. Recently, cyanobacteria have attracted great interest due to their crucial roles in global carbon and nitrogen cycles and their ability to produce clean and renewable biofuels. To survive in various environmental conditions, cyanobacteria have developed a complex signal transduction network to sense environmental signals and implement adaptive changes. The post-translational modifications (PTMs) systems play important regulatory roles in the signaling networks of cyanobacteria. The systematic investigation of PTMs could contribute to the comprehensive description of protein species and to elucidate potential biological roles of each protein species in cyanobacteria. Although the proteomic studies of PTMs carried out in cyanobacteria were limited, these data have provided clues to elucidate their sophisticated sensing mechanisms that contribute to their evolutionary and ecological success. This review aims to summarize the current status of PTM studies and recent publications regarding PTM proteomics in cyanobacteria, and discuss the novel developments and applications for the analysis of PTMs in cyanobacteria. Challenges, opportunities and future perspectives in the proteomics studies of PTMs in cyanobacteria are also discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Simultaneous Proteomic Discovery and Targeted Monitoring using Liquid Chromatography, Ion Mobility Spectrometry, and Mass Spectrometry*

PubMed Central

Burnum-Johnson, Kristin E.; Nie, Song; Casey, Cameron P.; Monroe, Matthew E.; Orton, Daniel J.; Ibrahim, Yehia M.; Gritsenko, Marina A.; Clauss, Therese R. W.; Shukla, Anil K.; Moore, Ronald J.; Purvine, Samuel O.; Shi, Tujin; Qian, Weijun; Liu, Tao; Baker, Erin S.; Smith, Richard D.

2016-01-01

Current proteomic approaches include both broad discovery measurements and quantitative targeted analyses. In many cases, discovery measurements are initially used to identify potentially important proteins (e.g. candidate biomarkers) and then targeted studies are employed to quantify a limited number of selected proteins. Both approaches, however, suffer from limitations. Discovery measurements aim to sample the whole proteome but have lower sensitivity, accuracy, and quantitation precision than targeted approaches, whereas targeted measurements are significantly more sensitive but only sample a limited portion of the proteome. Herein, we describe a new approach that performs both discovery and targeted monitoring (DTM) in a single analysis by combining liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled target peptides are spiked into tryptic digests and both the labeled and unlabeled peptides are detected using LC-IMS-MS instrumentation. Compared with the broad LC-MS discovery measurements, DTM yields greater peptide/protein coverage and detects lower abundance species. DTM also achieved detection limits similar to selected reaction monitoring (SRM) indicating its potential for combined high quality discovery and targeted analyses, which is a significant step toward the convergence of discovery and targeted approaches. PMID:27670688

Effects of conjugated linoleic acid (CLA) on fat accumulation, activity, and proteomics analysis in Caenorhabditis elegans.

PubMed

Shen, Peiyi; Kershaw, Jonathan C; Yue, Yiren; Wang, Ou; Kim, Kee-Hong; McClements, D Julian; Park, Yeonhwa

2018-05-30

Conjugated linoleic acid (CLA) has been reported to reduce fat storage in cell culture and animal models. In the current study, the effects of CLA on the fat accumulation, activities, and proteomics were investigated using Caenorhabditis elegans. 100 µM CLA-TG nanoemulsion significantly reduced fat accumulation by 29% compared to linoleic acid (LA)-TG treatment via sir-2.1 (the ortholog of Sirtuin 1), without altering the worm size, growth rate, and pumping rate of C. elegans. CLA significantly increased moving speed and amplitude (the average centroid displacement over the entire track) of wild type worms compared to the LA group and these effects were dependent on aak-2 (AMPKα ortholog) and sir-2.1. Proteomics analysis showed CLA treatment influences various proteins associated in reproduction and development, translation, metabolic processes, and catabolism and proteolysis, in C. elegans. We have also confirmed the proteomics data that CLA reduced the fat accumulation via abs-1 (ATP Synthase B homolog). However, there were no significant effects of CLA on brood size, progeny numbers, and hatchability compared to LA treatment. Copyright © 2018 Elsevier Ltd. All rights reserved.

A Proteomic View on the Role of Legume Symbiotic Interactions

PubMed Central

Larrainzar, Estíbaliz; Wienkoop, Stefanie

2017-01-01

Legume plants are key elements in sustainable agriculture and represent a significant source of plant-based protein for humans and animal feed worldwide. One specific feature of the family is the ability to establish nitrogen-fixing symbiosis with Rhizobium bacteria. Additionally, like most vascular flowering plants, legumes are able to form a mutualistic endosymbiosis with arbuscular mycorrhizal (AM) fungi. These beneficial associations can enhance the plant resistance to biotic and abiotic stresses. Understanding how symbiotic interactions influence and increase plant stress tolerance are relevant questions toward maintaining crop yield and food safety in the scope of climate change. Proteomics offers numerous tools for the identification of proteins involved in such responses, allowing the study of sub-cellular localization and turnover regulation, as well as the discovery of post-translational modifications (PTMs). The current work reviews the progress made during the last decades in the field of proteomics applied to the study of the legume-Rhizobium and -AM symbioses, and highlights their influence on the plant responses to pathogens and abiotic stresses. We further discuss future perspectives and new experimental approaches that are likely to have a significant impact on the field including peptidomics, mass spectrometric imaging, and quantitative proteomics. PMID:28769967

Contribution of proteomics to the study of plant pathogenic fungi.

PubMed

Gonzalez-Fernandez, Raquel; Jorrin-Novo, Jesus V

2012-01-01

Phytopathogenic fungi are one of the most damaging plant parasitic organisms, and can cause serious diseases and important yield losses in crops. The study of the biology of these microorganisms and the interaction with their hosts has experienced great advances in recent years due to the development of moderm, holistic and high-throughput -omic techniques, together with the increasing number of genome sequencing projects and the development of mutants and reverse genetics tools. We highlight among these -omic techniques the importance of proteomics, which has become a relevant tool in plant-fungus pathosystem research. Proteomics intends to identify gene products with a key role in pathogenicity and virulence. These studies would help in the search of key protein targets and in the development of agrochemicals, which may open new ways for crop disease diagnosis and protection. In this review, we made an overview on the contribution of proteomics to the knowledge of life cycle, infection mechanisms, and virulence of the plant pathogenic fungi. Data from current, innovative literature, according to both methodological and experimental systems, were summarized and discussed. Specific sections were devoted to the most studied fungal phytopathogens: Botrytis cinerea, Sclerotinia sclerotiorum, and Fusarium graminearum.

Shotgun proteomic analysis of Emiliania huxleyi, a marine phytoplankton species of major biogeochemical importance.

PubMed

Jones, Bethan M; Edwards, Richard J; Skipp, Paul J; O'Connor, C David; Iglesias-Rodriguez, M Debora

2011-06-01

Emiliania huxleyi is a unicellular marine phytoplankton species known to play a significant role in global biogeochemistry. Through the dual roles of photosynthesis and production of calcium carbonate (calcification), carbon is transferred from the atmosphere to ocean sediments. Almost nothing is known about the molecular mechanisms that control calcification, a process that is tightly regulated within the cell. To initiate proteomic studies on this important and phylogenetically remote organism, we have devised efficient protein extraction protocols and developed a bioinformatics pipeline that allows the statistically robust assignment of proteins from MS/MS data using preexisting EST sequences. The bioinformatics tool, termed BUDAPEST (Bioinformatics Utility for Data Analysis of Proteomics using ESTs), is fully automated and was used to search against data generated from three strains. BUDAPEST increased the number of identifications over standard protein database searches from 37 to 99 proteins when data were amalgamated. Proteins involved in diverse cellular processes were uncovered. For example, experimental evidence was obtained for a novel type I polyketide synthase and for various photosystem components. The proteomic and bioinformatic approaches developed in this study are of wider applicability, particularly to the oceanographic community where genomic sequence data for species of interest are currently scarce.

Impact of pyrrolidine-bispyrrole DNA minor groove binding agents and chirality on global proteomic profile in Escherichia Coli.

PubMed

Yang, Ya-Ting; Lin, Chun-Yu; Jeng, Jingyueh; Ong, Chi-Wi

2013-05-23

There is great interest in the design of small molecules that selectively target minor grooves of duplex DNA for controlling specific gene expression implicated in a disease. The design of chiral small molecules for rational drug design has attracted increasing attention due to the chirality of DNA. Yet, there is limited research on the chirality effect of minor groove binders on DNA interaction, especially at the protein expression level. This paper is an attempt to illustrate that DNA binding affinity might not provide a full picture on the biological activities. Drug interacting at the genomic level can be translated to the proteomic level. Here we have illustrated that although the chiral bispyrrole-pyrrolidine-oligoamides, PySSPy and PyRSPy, showed low binding affinity to DNA, their influence at the proteomic level is significant. More importantly, the chirality also plays a role. Two-dimensional proteomic profile to identify the differentially expressed protein in Escherichia coli DH5α (E coli DH5α) were investigated. E coli DH5α incubated with the chiral PySSPy and PyRSPy, diastereomeric at the pyrrolidine ring, showed differential expression of eighteen proteins as observed through two dimensional proteomic profiling. These eighteen proteins identified by MALDI_TOF/TOF MS include antioxidant defense, DNA protection, protein synthesis, chaperone, and stress response proteins. No statistically significant toxicity was observed at the tested drug concentrations as measured via MTT assay. The current results showed that the chiral PySSPy and PyRSPy impact on the proteomic profiling of E coli DH5α, implicating the importance of drug chirality on biological activities at the molecular level.

Forensic proteomics for the evaluation of the post-mortem decay in bones.

PubMed

Procopio, Noemi; Williams, Anna; Chamberlain, Andrew T; Buckley, Michael

2018-04-15

Current methods for evaluation the of post-mortem interval (PMI) of skeletal remains suffer from poor accuracy due to the great number of variables that affect the diagenetic process and to the lack of specific guidelines to address this issue. During decomposition, proteins can undergo cumulative decay over the time, resulting in a decrease in the range and abundance of proteins present (i.e., the proteome) in different tissues as well as in an increase of post-translational modifications occurring in these proteins. In this study, we investigate the applicability of bone proteomic analyses to simulated forensic contexts, looking for specific biomarkers that may help the estimation of PMI, as well as evaluate a previously discovered marker for the estimation of biological age. We noticed a reduction of particular plasma and muscle proteins with increasing PMIs, as well as an increased deamidation of biglycan, a protein with a role in modulating bone growth and mineralization. We also corroborated our previous results regarding the use of fetuin-A as a potential biomarker for the estimation of age-at-death, demonstrating the applicability and the great potential that proteomics may have towards forensic sciences. The estimation of the post-mortem interval has a key role in forensic investigations, however nowadays it still suffers from poor reliability, especially when body tissues are heavily decomposed. Here we propose for the first time the application of bone proteomics to the estimation of the time elapsed since death and found several new potential biomarkers to address this, demonstrating the applicability of proteomic analyses to forensic sciences. Copyright © 2018. Published by Elsevier B.V.

Proteomic Workflows for Biomarker Identification Using Mass Spectrometry — Technical and Statistical Considerations during Initial Discovery

PubMed Central

Orton, Dennis J.; Doucette, Alan A.

2013-01-01

Identification of biomarkers capable of differentiating between pathophysiological states of an individual is a laudable goal in the field of proteomics. Protein biomarker discovery generally employs high throughput sample characterization by mass spectrometry (MS), being capable of identifying and quantifying thousands of proteins per sample. While MS-based technologies have rapidly matured, the identification of truly informative biomarkers remains elusive, with only a handful of clinically applicable tests stemming from proteomic workflows. This underlying lack of progress is attributed in large part to erroneous experimental design, biased sample handling, as well as improper statistical analysis of the resulting data. This review will discuss in detail the importance of experimental design and provide some insight into the overall workflow required for biomarker identification experiments. Proper balance between the degree of biological vs. technical replication is required for confident biomarker identification. PMID:28250400

Integrated omics dissection of proteome dynamics during cardiac remodeling.

PubMed

Lau, Edward; Cao, Quan; Lam, Maggie P Y; Wang, Jie; Ng, Dominic C M; Bleakley, Brian J; Lee, Jessica M; Liem, David A; Wang, Ding; Hermjakob, Henning; Ping, Peipei

2018-01-09

Transcript abundance and protein abundance show modest correlation in many biological models, but how this impacts disease signature discovery in omics experiments is rarely explored. Here we report an integrated omics approach, incorporating measurements of transcript abundance, protein abundance, and protein turnover to map the landscape of proteome remodeling in a mouse model of pathological cardiac hypertrophy. Analyzing the hypertrophy signatures that are reproducibly discovered from each omics data type across six genetic strains of mice, we find that the integration of transcript abundance, protein abundance, and protein turnover data leads to 75% gain in discovered disease gene candidates. Moreover, the inclusion of protein turnover measurements allows discovery of post-transcriptional regulations across diverse pathways, and implicates distinct disease proteins not found in steady-state transcript and protein abundance data. Our results suggest that multi-omics investigations of proteome dynamics provide important insights into disease pathogenesis in vivo.

High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) for Mass Spectrometry-Based Proteomics

PubMed Central

Swearingen, Kristian E.; Moritz, Robert L.

2013-01-01

SUMMARY High field asymmetric waveform ion mobility spectrometry (FAIMS) is an atmospheric pressure ion mobility technique that separates gas-phase ions by their behavior in strong and weak electric fields. FAIMS is easily interfaced with electrospray ionization and has been implemented as an additional separation mode between liquid chromatography (LC) and mass spectrometry (MS) in proteomic studies. FAIMS separation is orthogonal to both LC and MS and is used as a means of on-line fractionation to improve detection of peptides in complex samples. FAIMS improves dynamic range and concomitantly the detection limits of ions by filtering out chemical noise. FAIMS can also be used to remove interfering ion species and to select peptide charge states optimal for identification by tandem MS. Here, we review recent developments in LC-FAIMS-MS and its application to MS-based proteomics. PMID:23194268

In-depth analysis of protein inference algorithms using multiple search engines and well-defined metrics.

PubMed

Audain, Enrique; Uszkoreit, Julian; Sachsenberg, Timo; Pfeuffer, Julianus; Liang, Xiao; Hermjakob, Henning; Sanchez, Aniel; Eisenacher, Martin; Reinert, Knut; Tabb, David L; Kohlbacher, Oliver; Perez-Riverol, Yasset

2017-01-06

In mass spectrometry-based shotgun proteomics, protein identifications are usually the desired result. However, most of the analytical methods are based on the identification of reliable peptides and not the direct identification of intact proteins. Thus, assembling peptides identified from tandem mass spectra into a list of proteins, referred to as protein inference, is a critical step in proteomics research. Currently, different protein inference algorithms and tools are available for the proteomics community. Here, we evaluated five software tools for protein inference (PIA, ProteinProphet, Fido, ProteinLP, MSBayesPro) using three popular database search engines: Mascot, X!Tandem, and MS-GF+. All the algorithms were evaluated using a highly customizable KNIME workflow using four different public datasets with varying complexities (different sample preparation, species and analytical instruments). We defined a set of quality control metrics to evaluate the performance of each combination of search engines, protein inference algorithm, and parameters on each dataset. We show that the results for complex samples vary not only regarding the actual numbers of reported protein groups but also concerning the actual composition of groups. Furthermore, the robustness of reported proteins when using databases of differing complexities is strongly dependant on the applied inference algorithm. Finally, merging the identifications of multiple search engines does not necessarily increase the number of reported proteins, but does increase the number of peptides per protein and thus can generally be recommended. Protein inference is one of the major challenges in MS-based proteomics nowadays. Currently, there are a vast number of protein inference algorithms and implementations available for the proteomics community. Protein assembly impacts in the final results of the research, the quantitation values and the final claims in the research manuscript. Even though protein inference is a crucial step in proteomics data analysis, a comprehensive evaluation of the many different inference methods has never been performed. Previously Journal of proteomics has published multiple studies about other benchmark of bioinformatics algorithms (PMID: 26585461; PMID: 22728601) in proteomics studies making clear the importance of those studies for the proteomics community and the journal audience. This manuscript presents a new bioinformatics solution based on the KNIME/OpenMS platform that aims at providing a fair comparison of protein inference algorithms (https://github.com/KNIME-OMICS). Six different algorithms - ProteinProphet, MSBayesPro, ProteinLP, Fido and PIA- were evaluated using the highly customizable workflow on four public datasets with varying complexities. Five popular database search engines Mascot, X!Tandem, MS-GF+ and combinations thereof were evaluated for every protein inference tool. In total >186 proteins lists were analyzed and carefully compare using three metrics for quality assessments of the protein inference results: 1) the numbers of reported proteins, 2) peptides per protein, and the 3) number of uniquely reported proteins per inference method, to address the quality of each inference method. We also examined how many proteins were reported by choosing each combination of search engines, protein inference algorithms and parameters on each dataset. The results show that using 1) PIA or Fido seems to be a good choice when studying the results of the analyzed workflow, regarding not only the reported proteins and the high-quality identifications, but also the required runtime. 2) Merging the identifications of multiple search engines gives almost always more confident results and increases the number of peptides per protein group. 3) The usage of databases containing not only the canonical, but also known isoforms of proteins has a small impact on the number of reported proteins. The detection of specific isoforms could, concerning the question behind the study, compensate for slightly shorter reports using the parsimonious reports. 4) The current workflow can be easily extended to support new algorithms and search engine combinations. Copyright © 2016. Published by Elsevier B.V.

Hypothalamus proteomics from mouse models with obesity and anorexia reveals therapeutic targets of appetite regulation

PubMed Central

Manousopoulou, A; Koutmani, Y; Karaliota, S; Woelk, C H; Manolakos, E S; Karalis, K; Garbis, S D

2016-01-01

Objective: This study examined the proteomic profile of the hypothalamus in mice exposed to a high-fat diet (HFD) or with the anorexia of acute illness. This comparison could provide insight on the effects of these two opposite states of energy balance on appetite regulation. Methods: Four to six-week-old male C56BL/6J mice were fed a normal (control 1 group; n=7) or a HFD (HFD group; n=10) for 8 weeks. The control 2 (n=7) and lipopolysaccharide (LPS) groups (n=10) were fed a normal diet for 8 weeks before receiving an injection of saline and LPS, respectively. Hypothalamic regions were analysed using a quantitative proteomics method based on a combination of techniques including iTRAQ stable isotope labeling, orthogonal two-dimensional liquid chromatography hyphenated with nanospray ionization and high-resolution mass spectrometry. Key proteins were validated with quantitative PCR. Results: Quantitative proteomics of the hypothalamous regions profiled a total of 9249 protein groups (q<0.05). Of these, 7718 protein groups were profiled with a minimum of two unique peptides for each. Hierachical clustering of the differentiated proteome revealed distinct proteomic signatures for the hypothalamus under the HFD and LPS nutritional conditions. Literature research with in silico bioinformatics interpretation of the differentiated proteome identified key biological relevant proteins and implicated pathways. Furthermore, the study identified potential pharmacologic targets. In the LPS groups, the anorexigen pro-opiomelanocortin was downregulated. In mice with obesity, nuclear factor-κB, glycine receptor subunit alpha-4 (GlyR) and neuropeptide Y levels were elevated, whereas serotonin receptor 1B levels decreased. Conclusions: High-precision quantitative proteomics revealed that under acute systemic inflammation in the hypothalamus as a response to LPS, homeostatic mechanisms mediating loss of appetite take effect. Conversely, under chronic inflammation in the hypothalamus as a response to HFD, mechanisms mediating a sustained ‘perpetual cycle' of appetite enhancement were observed. The GlyR protein may constitute a novel treatment target for the reduction of central orexigenic signals in obesity. PMID:27110685

Hypothalamus proteomics from mouse models with obesity and anorexia reveals therapeutic targets of appetite regulation.

PubMed

Manousopoulou, A; Koutmani, Y; Karaliota, S; Woelk, C H; Manolakos, E S; Karalis, K; Garbis, S D

2016-04-25

This study examined the proteomic profile of the hypothalamus in mice exposed to a high-fat diet (HFD) or with the anorexia of acute illness. This comparison could provide insight on the effects of these two opposite states of energy balance on appetite regulation. Four to six-week-old male C56BL/6J mice were fed a normal (control 1 group; n=7) or a HFD (HFD group; n=10) for 8 weeks. The control 2 (n=7) and lipopolysaccharide (LPS) groups (n=10) were fed a normal diet for 8 weeks before receiving an injection of saline and LPS, respectively. Hypothalamic regions were analysed using a quantitative proteomics method based on a combination of techniques including iTRAQ stable isotope labeling, orthogonal two-dimensional liquid chromatography hyphenated with nanospray ionization and high-resolution mass spectrometry. Key proteins were validated with quantitative PCR. Quantitative proteomics of the hypothalamous regions profiled a total of 9249 protein groups (q<0.05). Of these, 7718 protein groups were profiled with a minimum of two unique peptides for each. Hierachical clustering of the differentiated proteome revealed distinct proteomic signatures for the hypothalamus under the HFD and LPS nutritional conditions. Literature research with in silico bioinformatics interpretation of the differentiated proteome identified key biological relevant proteins and implicated pathways. Furthermore, the study identified potential pharmacologic targets. In the LPS groups, the anorexigen pro-opiomelanocortin was downregulated. In mice with obesity, nuclear factor-κB, glycine receptor subunit alpha-4 (GlyR) and neuropeptide Y levels were elevated, whereas serotonin receptor 1B levels decreased. High-precision quantitative proteomics revealed that under acute systemic inflammation in the hypothalamus as a response to LPS, homeostatic mechanisms mediating loss of appetite take effect. Conversely, under chronic inflammation in the hypothalamus as a response to HFD, mechanisms mediating a sustained 'perpetual cycle' of appetite enhancement were observed. The GlyR protein may constitute a novel treatment target for the reduction of central orexigenic signals in obesity.

Sma3s: A universal tool for easy functional annotation of proteomes and transcriptomes.

PubMed

Casimiro-Soriguer, Carlos S; Muñoz-Mérida, Antonio; Pérez-Pulido, Antonio J

2017-06-01

The current cheapening of next-generation sequencing has led to an enormous growth in the number of sequenced genomes and transcriptomes, allowing wet labs to get the sequences from their organisms of study. To make the most of these data, one of the first things that should be done is the functional annotation of the protein-coding genes. But it used to be a slow and tedious step that can involve the characterization of thousands of sequences. Sma3s is an accurate computational tool for annotating proteins in an unattended way. Now, we have developed a completely new version, which includes functionalities that will be of utility for fundamental and applied science. Currently, the results provide functional categories such as biological processes, which become useful for both characterizing particular sequence datasets and comparing results from different projects. But one of the most important implemented innovations is that it has now low computational requirements, and the complete annotation of a simple proteome or transcriptome usually takes around 24 hours in a personal computer. Sma3s has been tested with a large amount of complete proteomes and transcriptomes, and it has demonstrated its potential in health science and other specific projects. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Covering complete proteomes with X-ray structures: A current snapshot

DOE PAGES

Mizianty, Marcin J.; Fan, Xiao; Yan, Jing; ...

2014-10-23

Structural genomics programs have developed and applied structure-determination pipelines to a wide range of protein targets, facilitating the visualization of macromolecular interactions and the understanding of their molecular and biochemical functions. The fundamental question of whether three-dimensional structures of all proteins and all functional annotations can be determined using X-ray crystallography is investigated. A first-of-its-kind large-scale analysis of crystallization propensity for all proteins encoded in 1953 fully sequenced genomes was performed. It is shown that current X-ray crystallographic knowhow combined with homology modeling can provide structures for 25% of modeling families (protein clusters for which structural models can be obtainedmore » through homology modeling), with at least one structural model produced for each Gene Ontology functional annotation. The coverage varies between superkingdoms, with 19% for eukaryotes, 35% for bacteria and 49% for archaea, and with those of viruses following the coverage values of their hosts. It is shown that the crystallization propensities of proteomes from the taxonomic superkingdoms are distinct. The use of knowledge-based target selection is shown to substantially increase the ability to produce X-ray structures. It is demonstrated that the human proteome has one of the highest attainable coverage values among eukaryotes, and GPCR membrane proteins suitable for X-ray structure determination were determined.« less

Spermatogenesis in mammals: proteomic insights.

PubMed

Chocu, Sophie; Calvel, Pierre; Rolland, Antoine D; Pineau, Charles

2012-08-01

Spermatogenesis is a highly sophisticated process involved in the transmission of genetic heritage. It includes halving ploidy, repackaging of the chromatin for transport, and the equipment of developing spermatids and eventually spermatozoa with the advanced apparatus (e.g., tightly packed mitochondrial sheat in the mid piece, elongating of the tail, reduction of cytoplasmic volume) to elicit motility once they reach the epididymis. Mammalian spermatogenesis is divided into three phases. In the first the primitive germ cells or spermatogonia undergo a series of mitotic divisions. In the second the spermatocytes undergo two consecutive divisions in meiosis to produce haploid spermatids. In the third the spermatids differentiate into spermatozoa in a process called spermiogenesis. Paracrine, autocrine, juxtacrine, and endocrine pathways all contribute to the regulation of the process. The array of structural elements and chemical factors modulating somatic and germ cell activity is such that the network linking the various cellular activities during spermatogenesis is unimaginably complex. Over the past two decades, advances in genomics have greatly improved our knowledge of spermatogenesis, by identifying numerous genes essential for the development of functional male gametes. Large-scale analyses of testicular function have deepened our insight into normal and pathological spermatogenesis. Progress in genome sequencing and microarray technology have been exploited for genome-wide expression studies, leading to the identification of hundreds of genes differentially expressed within the testis. However, although proteomics has now come of age, the proteomics-based investigation of spermatogenesis remains in its infancy. Here, we review the state-of-the-art of large-scale proteomic analyses of spermatogenesis, from germ cell development during sex determination to spermatogenesis in the adult. Indeed, a few laboratories have undertaken differential protein profiling expression studies and/or systematic analyses of testicular proteomes in entire organs or isolated cells from various species. We consider the pros and cons of proteomics for studying the testicular germ cell gene expression program. Finally, we address the use of protein datasets, through integrative genomics (i.e., combining genomics, transcriptomics, and proteomics), bioinformatics, and modelling.

Megadalton Complexes in the Chloroplast Stroma of Arabidopsis thaliana Characterized by Size Exclusion Chromatography, Mass Spectrometry, and Hierarchical Clustering*

PubMed Central

Olinares, Paul Dominic B.; Ponnala, Lalit; van Wijk, Klaas J.

2010-01-01

To characterize MDa-sized macromolecular chloroplast stroma protein assemblies and to extend coverage of the chloroplast stroma proteome, we fractionated soluble chloroplast stroma in the non-denatured state by size exclusion chromatography with a size separation range up to ∼5 MDa. To maximize protein complex stability and resolution of megadalton complexes, ionic strength and composition were optimized. Subsequent high accuracy tandem mass spectrometry analysis (LTQ-Orbitrap) identified 1081 proteins across the complete native mass range. Protein complexes and assembly states above 0.8 MDa were resolved using hierarchical clustering, and protein heat maps were generated from normalized protein spectral counts for each of the size exclusion chromatography fractions; this complemented previous analysis of stromal complexes up to 0.8 MDa (Peltier, J. B., Cai, Y., Sun, Q., Zabrouskov, V., Giacomelli, L., Rudella, A., Ytterberg, A. J., Rutschow, H., and van Wijk, K. J. (2006) The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts. Mol. Cell. Proteomics 5, 114–133). This combined experimental and bioinformatics analyses resolved chloroplast ribosomes in different assembly and functional states (e.g. 30, 50, and 70 S), which enabled the identification of plastid homologues of prokaryotic ribosome assembly factors as well as proteins involved in co-translational modifications, targeting, and folding. The roles of these ribosome-associating proteins will be discussed. Known RNA splice factors (e.g. CAF1/WTF1/RNC1) as well as uncharacterized proteins with RNA-binding domains (pentatricopeptide repeat, RNA recognition motif, and chloroplast ribosome maturation), RNases, and DEAD box helicases were found in various sized complexes. Chloroplast DNA (>3 MDa) was found in association with the complete heteromeric plastid-encoded DNA polymerase complex, and a dozen other DNA-binding proteins, e.g. DNA gyrase, topoisomerase, and various DNA repair enzymes. The heteromeric ≥5-MDa pyruvate dehydrogenase complex and the 0.8–1-MDa acetyl-CoA carboxylase complex associated with uncharacterized biotin carboxyl carrier domain proteins constitute the entry point to fatty acid metabolism in leaves; we suggest that their large size relates to the need for metabolic channeling. Protein annotations and identification data are available through the Plant Proteomics Database, and mass spectrometry data are available through Proteomics Identifications database. PMID:20423899

Redox Proteomics Applied to the Thiol Secretome.

PubMed

Ghezzi, Pietro; Chan, Philippe

2017-03-01

Secreted proteins are important both as signaling molecules and potential biomarkers. Recent Advances: Protein can undergo different types of oxidation, both in physiological conditions or under oxidative stress. Several redox proteomics techniques have been successfully applied to the identification of glutathionylated proteins, an oxidative post-translational modification consisting in the formation of a mixed disulfide between a protein cysteine and glutathione. Redox proteomics has also been used to study other forms of protein oxidation. Because of the highest proportion of free cysteines in the cytosol, redox proteomics of protein thiols has focused, so far, on intracellular proteins. However, plasma proteins, such as transthyretin and albumin, have been described as glutathionylated or cysteinylated. The present review discusses the redox state of protein cysteines in relation to their cellular distribution. We describe the various approaches used to detect secreted glutathionylated proteins, the only thiol modification studied so far in secreted proteins, and the specific problems presented in the study of the secretome. This review focusses on glutathionylated proteins secreted under inflammatory conditions and that may act as soluble mediators (cytokines). Future studies on the redox secretome (including other forms of oxidation) might identify new soluble mediators and biomarkers of oxidative stress. Antioxid. Redox Signal. 26, 299-312.

Optimization of quantitative proteomic analysis of clots generated from plasma of patients with venous thromboembolism.

PubMed

Stachowicz, Aneta; Siudut, Jakub; Suski, Maciej; Olszanecki, Rafał; Korbut, Ryszard; Undas, Anetta; Wiśniewski, Jacek R

2017-01-01

It is well known that fibrin network binds a large variety of proteins, including inhibitors and activators of fibrinolysis, which may affect clot properties, such as stability and susceptibility to fibrinolysis. Specific plasma clot composition differs between individuals and may change in disease states. However, the plasma clot proteome has not yet been in-depth analyzed, mainly due to technical difficulty related to the presence of a highly abundant protein-fibrinogen and fibrin that forms a plasma clot. The aim of our study was to optimize quantitative proteomic analysis of fibrin clots prepared ex vivo from citrated plasma of the peripheral blood drawn from patients with prior venous thromboembolism (VTE). We used a multiple enzyme digestion filter aided sample preparation, a multienzyme digestion (MED) FASP method combined with LC-MS/MS analysis performed on a Proxeon Easy-nLC System coupled to the Q Exactive HF mass spectrometer. We also evaluated the impact of peptide fractionation with pipet-tip strong anion exchange (SAX) method on the obtained results. Our proteomic approach revealed 476 proteins repeatedly identified in the plasma fibrin clots from patients with VTE including extracellular vesicle-derived proteins, lipoproteins, fibrinolysis inhibitors, and proteins involved in immune responses. The MED FASP method using three different enzymes: LysC, trypsin and chymotrypsin increased the number of identified peptides and proteins and their sequence coverage as compared to a single step digestion. Peptide fractionation with a pipet-tip strong anion exchange (SAX) protocol increased the depth of proteomic analyses, but also extended the time needed for sample analysis with LC-MS/MS. The MED FASP method combined with a label-free quantification is an excellent proteomic approach for the analysis of fibrin clots prepared ex vivo from citrated plasma of patients with prior VTE.

Quantitative proteomic characterization of the lung extracellular matrix in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.

PubMed

Åhrman, Emma; Hallgren, Oskar; Malmström, Lars; Hedström, Ulf; Malmström, Anders; Bjermer, Leif; Zhou, Xiao-Hong; Westergren-Thorsson, Gunilla; Malmström, Johan

2018-03-01

Remodeling of the extracellular matrix (ECM) is a common feature in lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Here, we applied a sequential tissue extraction strategy to describe disease-specific remodeling of human lung tissue in disease, using end-stages of COPD and IPF. Our strategy was based on quantitative comparison of the disease proteomes, with specific focus on the matrisome, using data-independent acquisition and targeted data analysis (SWATH-MS). Our work provides an in-depth proteomic characterization of human lung tissue during impaired tissue remodeling. In addition, we show important quantitative and qualitative effects of the solubility of matrisome proteins. COPD was characterized by a disease-specific increase in ECM regulators, metalloproteinase inhibitor 3 (TIMP3) and matrix metalloproteinase 28 (MMP-28), whereas for IPF, impairment in cell adhesion proteins, such as collagen VI and laminins, was most prominent. For both diseases, we identified increased levels of proteins involved in the regulation of endopeptidase activity, with several proteins belonging to the serpin family. The established human lung quantitative proteome inventory and the construction of a tissue-specific protein assay library provides a resource for future quantitative proteomic analyses of human lung tissues. We present a sequential tissue extraction strategy to determine changes in extractability of matrisome proteins in end-stage COPD and IPF compared to healthy control tissue. Extensive quantitative analysis of the proteome changes of the disease states revealed altered solubility of matrisome proteins involved in ECM regulators and cell-ECM communication. The results highlight disease-specific remodeling mechanisms associated with COPD and IPF. Copyright © 2018 Elsevier B.V. All rights reserved.

Gene and protein expression following exposure to radiofrequency fields from mobile phones.

PubMed

Vanderstraeten, Jacques; Verschaeve, Luc

2008-09-01

Since 1999, several articles have been published on genome-wide and/or proteome-wide response after exposure to radiofrequency (RF) fields whose signal and intensities were similar to or typical of those of currently used mobile telephones. These studies were performed using powerful high-throughput screening techniques (HTSTs) of transcriptomics and/or proteomics, which allow for the simultaneous screening of the expression of thousands of genes or proteins. We reviewed these HTST-based studies and compared the results with currently accepted concepts about the effects of RF fields on gene expression. In this article we also discuss these last in light of the recent concept of microwave-assisted chemistry. To date, the results of HTST-based studies of transcriptomics and/or proteomics after exposure to RF fields relevant to human exposure are still inconclusive, as most of the positive reports are flawed by methodologic imperfections or shortcomings. In addition, when positive findings were reported, no precise response pattern could be identified in a reproducible way. In particular, results from HTST studies tend to exclude the role of a cell stressor for exposure to RF fields at nonthermal intensities. However, on the basis of lessons from microwave-assisted chemistry, we can assume that RF fields might affect heat-sensitive gene or protein expression to an extent larger than would be predicted from temperature change only. But in all likelihood, this would concern intensities higher than those relevant to usual human exposure. The precise role of transcriptomics and proteomics in the screening of bioeffects from exposure to RF fields from mobile phones is still uncertain in view of the lack of positively identified phenotypic change and the lack of theoretical, as well as experimental, arguments for specific gene and/or protein response patterns after this kind of exposure.

Korea University Joins the International Efforts in Clinical Proteogenomics Cancer Research | Office of Cancer Clinical Proteomics Research

Cancer.gov

The National Cancer Institute's (NCI) Office of Cancer Clinical Proteomics Research, part of the National Institutes of Health, and Korea University (KU) located in The Republic of Korea have signed a Memorandum of Understanding (MOU) in clinical proteogenomics cancer research. The MOU between NCI and KU represents the twenty-ninth institution and eleventh country to join the continued effort of the International Cancer Proteogenome Consortium (ICPC), an effort catalyzed through the vision of the 47th Vice President of the United States Joseph R. Biden, Jr. and the Cancer Moonshot.

Toxoplasmosis

DTIC Science & Technology

2011-06-01

Sarcocystis sp, which also can mimic T. gondii, form sarcocysts that contain various stages of the parasite (Figs 12.90 &12.91), including mature, banana ... Flowers SA. Global protein expression analysis in apicomplexan parasites: current status. Proteomics. 2005;5:918-924. Acknowledgements The authors

Deterministic protein inference for shotgun proteomics data provides new insights into Arabidopsis pollen development and function

PubMed Central

Grobei, Monica A.; Qeli, Ermir; Brunner, Erich; Rehrauer, Hubert; Zhang, Runxuan; Roschitzki, Bernd; Basler, Konrad; Ahrens, Christian H.; Grossniklaus, Ueli

2009-01-01

Pollen, the male gametophyte of flowering plants, represents an ideal biological system to study developmental processes, such as cell polarity, tip growth, and morphogenesis. Upon hydration, the metabolically quiescent pollen rapidly switches to an active state, exhibiting extremely fast growth. This rapid switch requires relevant proteins to be stored in the mature pollen, where they have to retain functionality in a desiccated environment. Using a shotgun proteomics approach, we unambiguously identified ∼3500 proteins in Arabidopsis pollen, including 537 proteins that were not identified in genetic or transcriptomic studies. To generate this comprehensive reference data set, which extends the previously reported pollen proteome by a factor of 13, we developed a novel deterministic peptide classification scheme for protein inference. This generally applicable approach considers the gene model–protein sequence–protein accession relationships. It allowed us to classify and eliminate ambiguities inherently associated with any shotgun proteomics data set, to report a conservative list of protein identifications, and to seamlessly integrate data from previous transcriptomics studies. Manual validation of proteins unambiguously identified by a single, information-rich peptide enabled us to significantly reduce the false discovery rate, while keeping valuable identifications of shorter and lower abundant proteins. Bioinformatic analyses revealed a higher stability of pollen proteins compared to those of other tissues and implied a protein family of previously unknown function in vesicle trafficking. Interestingly, the pollen proteome is most similar to that of seeds, indicating physiological similarities between these developmentally distinct tissues. PMID:19546170

Less is More: Membrane Protein Digestion Beyond Urea-Trypsin Solution for Next-level Proteomics.

PubMed

Zhang, Xi

2015-09-01

The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea-trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting-not destroying-structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteomics of exhaled breath: methodological nuances and pitfalls.

PubMed

Kurova, Viktoria S; Anaev, Eldar C; Kononikhin, Alexey S; Fedorchenko, Kristina Yu; Popov, Igor A; Kalupov, Timothey L; Bratanov, Dmitriy O; Nikolaev, Eugenie N; Varfolomeev, Sergey D

2009-01-01

The analysis of exhaled breath condensate (EBC) can be an alternative to traditional endoscopic sampling of lower respiratory tract secretions. This is a simple non-invasive method of diagnosing respiratory diseases, in particular, respiratory inflammatory processes. Samples were collected with a special device-condenser (ECoScreen, VIASYS Healthcare, Germany), then treated with trypsin according to the proteomics protocol for standard protein mixtures and analyzed by nanoflow high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) with a 7-Tesla Finnigan LTQ-FT mass spectrometer (Thermo Electron, Germany). Mascot software (Matrixscience) was used for screening the database NCBInr for proteins corresponding to the peptide maps that were obtained. EBCs from 17 young healthy non-smoking donors were collected. Different methods for concentrating protein were compared in order to optimize EBC preparations for proteomic analysis. The procedure that was chosen allowed identification of proteins exhaled by healthy people. The major proteins in the condensates were cytoskeletal keratins. Another 12 proteins were identified in EBC from healthy non-smokers. Some keratins were found in the ambient air and may be considered exogenous components of exhaled air. Knowledge of the normal proteome of exhaled breath allows one to look for biomarkers of different disease states in EBC. Proteins in ambient air can be identified in the respiratory tract and should be excluded from the analysis of the proteome of EBC. The results obtained allowed us to choose the most effective procedure of sample preparation when working with samples containing very low protein concentrations.

Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

PubMed

Pollock, Samuel B; Hu, Amy; Mou, Yun; Martinko, Alexander J; Julien, Olivier; Hornsby, Michael; Ploder, Lynda; Adams, Jarrett J; Geng, Huimin; Müschen, Markus; Sidhu, Sachdev S; Moffat, Jason; Wells, James A

2018-03-13

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states. Copyright © 2018 the Author(s). Published by PNAS.

NMR approaches in structure-based lead discovery: Recent developments and new frontiers for targeting multi-protein complexes

PubMed Central

Dias, David M.; Ciulli, Alessio

2014-01-01

Nuclear magnetic resonance (NMR) spectroscopy is a pivotal method for structure-based and fragment-based lead discovery because it is one of the most robust techniques to provide information on protein structure, dynamics and interaction at an atomic level in solution. Nowadays, in most ligand screening cascades, NMR-based methods are applied to identify and structurally validate small molecule binding. These can be high-throughput and are often used synergistically with other biophysical assays. Here, we describe current state-of-the-art in the portfolio of available NMR-based experiments that are used to aid early-stage lead discovery. We then focus on multi-protein complexes as targets and how NMR spectroscopy allows studying of interactions within the high molecular weight assemblies that make up a vast fraction of the yet untargeted proteome. Finally, we give our perspective on how currently available methods could build an improved strategy for drug discovery against such challenging targets. PMID:25175337

Omics-based biomarkers: current status and potential use in the clinic.

PubMed

Quezada, Héctor; Guzmán-Ortiz, Ana Laura; Díaz-Sánchez, Hugo; Valle-Rios, Ricardo; Aguirre-Hernández, Jesús

In recent years, the use of high-throughput omics technologies has led to the rapid discovery of many candidate biomarkers. However, few of them have made the transition to the clinic. In this review, the promise of omics technologies to contribute to the process of biomarker development is described. An overview of the current state in this area is presented with examples of genomics, proteomics, transcriptomics, metabolomics and microbiomics biomarkers in the field of oncology, along with some proposed strategies to accelerate their validation and translation to improve the care of patients with neoplasms. The inherent complexity underlying neoplasms combined with the requirement of developing well-designed biomarker discovery processes based on omics technologies present a challenge for the effective development of biomarkers that may be useful in guiding therapies, addressing disease risks, and predicting clinical outcomes. Copyright © 2017 Hospital Infantil de México Federico Gómez. Publicado por Masson Doyma México S.A. All rights reserved.

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.

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

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

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

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

Hydrophobic Interaction Chromatography for Bottom-Up Proteomics Analysis of Single Proteins and Protein Complexes.

PubMed

Rackiewicz, Michal; Große-Hovest, Ludger; Alpert, Andrew J; Zarei, Mostafa; Dengjel, Jörn

2017-06-02

Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used to study post-translational modifications of proteins and drug-protein interactions. In the current manuscript we employed HIC to separate proteins, followed by bottom-up LC-MS/MS experiments. We used this approach to fractionate antibody species followed by comprehensive peptide mapping as well as to study protein complexes in human cells. HIC-reversed-phase chromatography (RPC)-mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.

Beyond the Natural Proteome: Nondegenerate Saturation Mutagenesis-Methodologies and Advantages.

PubMed

Ferreira Amaral, M M; Frigotto, L; Hine, A V

2017-01-01

Beyond the natural proteome, high-throughput mutagenesis offers the protein engineer an opportunity to "tweak" the wild-type activity of a protein to create a recombinant protein with required attributes. Of the various approaches available, saturation mutagenesis is one of the core techniques employed by protein engineers, and in recent times, nondegenerate saturation mutagenesis is emerging as the approach of choice. This review compares the current methodologies available for conducting nondegenerate saturation mutagenesis with traditional, degenerate saturation and briefly outlines the options available for screening the resulting libraries, to discover a novel protein with the required activity and/or specificity. © 2017 Elsevier Inc. All rights reserved.

Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

Cancer.gov

The NCI CPTAC program is applying its expertise in quantitative proteomics to develop assays for RAS pathway proteins. Targets include key phosphopeptides that should increase our understanding of how the RAS pathway is regulated.

Detection of Elevated Plasma Levels of EGF Receptor Prior to Breast Cancer Diagnosis among Hormone Therapy Users

PubMed Central

Pitteri, Sharon J.; Amon, Lynn M.; Buson, Tina Busald; Zhang, Yuzheng; Johnson, Melissa M.; Chin, Alice; Kennedy, Jacob; Wong, Chee-Hong; Zhang, Qing; Wang, Hong; Lampe, Paul D.; Prentice, Ross L.; McIntosh, Martin W.; Hanash, Samir M.; Li, Christopher I.

2010-01-01

Applying advanced proteomic technologies to prospectively collected specimens from large studies is one means of identifying preclinical changes in plasma proteins that are potentially relevant to the early detection of diseases like breast cancer. We conducted fourteen independent quantitative proteomics experiments comparing pooled plasma samples collected from 420 estrogen receptor positive (ER+) breast cancer patients ≤17 months prior to their diagnosis and matched controls. Based on the over 3.4 million tandem mass spectra collected in the discovery set, 503 proteins were quantified of which 57 differentiated cases from controls with a p-value<0.1. Seven of these proteins, for which quantitative ELISA assays were available, were assessed in an independent validation set. Of these candidates, epidermal growth factor receptor (EGFR) was validated as a predictor of breast cancer risk in an independent set of preclinical plasma samples for women overall [odds ratio (OR)=1.44, p-value=0.0008], and particularly for current users of estrogen plus progestin (E+P) menopausal hormone therapy (OR=2.49, p-value=0.0001). Among current E+P users EGFR's sensitivity for breast cancer risk was 31% with 90% specificity. While EGFR's sensitivity and specificity are insufficient for a clinically useful early detection biomarker, this study suggests that proteins that are elevated preclinically in women who go on to develop breast cancer can be discovered and validated using current proteomic technologies. Further studies are warranted to both examine the role of EGFR and to discover and validate other proteins that could potentially be used for breast cancer early detection. PMID:20959476

Structure and function of nucleotide sugar transporters: Current progress.

PubMed

Hadley, Barbara; Maggioni, Andrea; Ashikov, Angel; Day, Christopher J; Haselhorst, Thomas; Tiralongo, Joe

2014-06-01

The proteomes of eukaryotes, bacteria and archaea are highly diverse due, in part, to the complex post-translational modification of protein glycosylation. The diversity of glycosylation in eukaryotes is reliant on nucleotide sugar transporters to translocate specific nucleotide sugars that are synthesised in the cytosol and nucleus, into the endoplasmic reticulum and Golgi apparatus where glycosylation reactions occur. Thirty years of research utilising multidisciplinary approaches has contributed to our current understanding of NST function and structure. In this review, the structure and function, with reference to various disease states, of several NSTs including the UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, GDP-fucose, UDP-N-acetylglucosamine/UDP-glucose/GDP-mannose and CMP-sialic acid transporters will be described. Little is known regarding the exact structure of NSTs due to difficulties associated with crystallising membrane proteins. To date, no three-dimensional structure of any NST has been elucidated. What is known is based on computer predictions, mutagenesis experiments, epitope-tagging studies, in-vitro assays and phylogenetic analysis. In this regard the best-characterised NST to date is the CMP-sialic acid transporter (CST). Therefore in this review we will provide the current state-of-play with respect to the structure-function relationship of the (CST). In particular we have summarised work performed by a number groups detailing the affect of various mutations on CST transport activity, efficiency, and substrate specificity.

Dynamic proteomics emphasizes the importance of selective mRNA translation and protein turnover during Arabidopsis seed germination.

PubMed

Galland, Marc; Huguet, Romain; Arc, Erwann; Cueff, Gwendal; Job, Dominique; Rajjou, Loïc

2014-01-01

During seed germination, the transition from a quiescent metabolic state in a dry mature seed to a proliferative metabolic state in a vigorous seedling is crucial for plant propagation as well as for optimizing crop yield. This work provides a detailed description of the dynamics of protein synthesis during the time course of germination, demonstrating that mRNA translation is both sequential and selective during this process. The complete inhibition of the germination process in the presence of the translation inhibitor cycloheximide established that mRNA translation is critical for Arabidopsis seed germination. However, the dynamics of protein turnover and the selectivity of protein synthesis (mRNA translation) during Arabidopsis seed germination have not been addressed yet. Based on our detailed knowledge of the Arabidopsis seed proteome, we have deepened our understanding of seed mRNA translation during germination by combining two-dimensional gel-based proteomics with dynamic radiolabeled proteomics using a radiolabeled amino acid precursor, namely [(35)S]-methionine, in order to highlight de novo protein synthesis, stability, and turnover. Our data confirm that during early imbibition, the Arabidopsis translatome keeps reflecting an embryonic maturation program until a certain developmental checkpoint. Furthermore, by dividing the seed germination time lapse into discrete time windows, we highlight precise and specific patterns of protein synthesis. These data refine and deepen our knowledge of the three classical phases of seed germination based on seed water uptake during imbibition and reveal that selective mRNA translation is a key feature of seed germination. Beyond the quantitative control of translational activity, both the selectivity of mRNA translation and protein turnover appear as specific regulatory systems, critical for timing the molecular events leading to successful germination and seedling establishment.

Dynamic Proteomics Emphasizes the Importance of Selective mRNA Translation and Protein Turnover during Arabidopsis Seed Germination*

PubMed Central

Galland, Marc; Huguet, Romain; Arc, Erwann; Cueff, Gwendal; Job, Dominique; Rajjou, Loïc

2014-01-01

During seed germination, the transition from a quiescent metabolic state in a dry mature seed to a proliferative metabolic state in a vigorous seedling is crucial for plant propagation as well as for optimizing crop yield. This work provides a detailed description of the dynamics of protein synthesis during the time course of germination, demonstrating that mRNA translation is both sequential and selective during this process. The complete inhibition of the germination process in the presence of the translation inhibitor cycloheximide established that mRNA translation is critical for Arabidopsis seed germination. However, the dynamics of protein turnover and the selectivity of protein synthesis (mRNA translation) during Arabidopsis seed germination have not been addressed yet. Based on our detailed knowledge of the Arabidopsis seed proteome, we have deepened our understanding of seed mRNA translation during germination by combining two-dimensional gel-based proteomics with dynamic radiolabeled proteomics using a radiolabeled amino acid precursor, namely [35S]-methionine, in order to highlight de novo protein synthesis, stability, and turnover. Our data confirm that during early imbibition, the Arabidopsis translatome keeps reflecting an embryonic maturation program until a certain developmental checkpoint. Furthermore, by dividing the seed germination time lapse into discrete time windows, we highlight precise and specific patterns of protein synthesis. These data refine and deepen our knowledge of the three classical phases of seed germination based on seed water uptake during imbibition and reveal that selective mRNA translation is a key feature of seed germination. Beyond the quantitative control of translational activity, both the selectivity of mRNA translation and protein turnover appear as specific regulatory systems, critical for timing the molecular events leading to successful germination and seedling establishment. PMID:24198433

Precision medicine for psychopharmacology: a general introduction.

PubMed

Shin, Cheolmin; Han, Changsu; Pae, Chi-Un; Patkar, Ashwin A

2016-07-01

Precision medicine is an emerging medical model that can provide accurate diagnoses and tailored therapeutic strategies for patients based on data pertaining to genes, microbiomes, environment, family history and lifestyle. Here, we provide basic information about precision medicine and newly introduced concepts, such as the precision medicine ecosystem and big data processing, and omics technologies including pharmacogenomics, pharamacometabolomics, pharmacoproteomics, pharmacoepigenomics, connectomics and exposomics. The authors review the current state of omics in psychiatry and the future direction of psychopharmacology as it moves towards precision medicine. Expert commentary: Advances in precision medicine have been facilitated by achievements in multiple fields, including large-scale biological databases, powerful methods for characterizing patients (such as genomics, proteomics, metabolomics, diverse cellular assays, and even social networks and mobile health technologies), and computer-based tools for analyzing large amounts of data.

The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components

PubMed Central

Fischer, Martina; Jehmlich, Nico; Rose, Laura; Koch, Sophia; Laue, Michael; Renard, Bernhard Y.; Schmidt, Frank; Heuer, Dagmar

2015-01-01

Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells’ antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection. PMID:26042774

Proteome Speciation by Mass Spectrometry: Characterization of Composite Protein Mixtures in Milk Replacers.

PubMed

Gaspari, Marco; Chiesa, Luca; Nicastri, Annalisa; Gabriele, Caterina; Harper, Valeria; Britti, Domenico; Cuda, Giovanni; Procopio, Antonio

2016-12-06

The ability of tandem mass spectrometry to determine the primary structure of proteolytic peptides can be exploited to trace back the organisms from which the corresponding proteins were extracted. This information can be important when food products, such as protein powders, can be supplemented with lower-quality starting materials. In order to dissect the origin of proteinaceous material composing a given unknown mixture, a two-step database search strategy for bottom-up nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) data was implemented. A single nanoLC-MS/MS analysis was sufficient not only to determine the qualitative composition of the mixtures under examination, but also to assess the relative percent composition of the various proteomes, if dedicated calibration curves were previously generated. The approach of two-step database search for qualitative analysis and proteome total ion current (pTIC) calculation for quantitative analysis was applied to several binary and ternary mixtures which mimic the composition of milk replacers typically used in calf feeding.

Proteomic evaluation of genetically modified crops: current status and challenges

PubMed Central

Gong, Chun Yan; Wang, Tai

2013-01-01

Hectares of genetically modified (GM) crops have increased exponentially since 1996, when such crops began to be commercialized. GM biotechnology, together with conventional breeding, has become the main approach to improving agronomic traits of crops. However, people are concerned about the safety of GM crops, especially GM-derived food and feed. Many efforts have been made to evaluate the unintended effects caused by the introduction of exogenous genes. “Omics” techniques have advantages over targeted analysis in evaluating such crops because of their use of high-throughput screening. Proteins are key players in gene function and are directly involved in metabolism and cellular development or have roles as toxins, antinutrients, or allergens, which are essential for human health. Thus, proteomics can be expected to become one of the most useful tools in safety assessment. This review assesses the potential of proteomics in evaluating various GM crops. We further describe the challenges in ensuring homogeneity and sensitivity in detection techniques. PMID:23471542

A large synthetic peptide and phosphopeptide reference library for mass spectrometry-based proteomics.

PubMed

Marx, Harald; Lemeer, Simone; Schliep, Jan Erik; Matheron, Lucrece; Mohammed, Shabaz; Cox, Jürgen; Mann, Matthias; Heck, Albert J R; Kuster, Bernhard

2013-06-01

We present a peptide library and data resource of >100,000 synthetic, unmodified peptides and their phosphorylated counterparts with known sequences and phosphorylation sites. Analysis of the library by mass spectrometry yielded a data set that we used to evaluate the merits of different search engines (Mascot and Andromeda) and fragmentation methods (beam-type collision-induced dissociation (HCD) and electron transfer dissociation (ETD)) for peptide identification. We also compared the sensitivities and accuracies of phosphorylation-site localization tools (Mascot Delta Score, PTM score and phosphoRS), and we characterized the chromatographic behavior of peptides in the library. We found that HCD identified more peptides and phosphopeptides than did ETD, that phosphopeptides generally eluted later from reversed-phase columns and were easier to identify than unmodified peptides and that current computational tools for proteomics can still be substantially improved. These peptides and spectra will facilitate the development, evaluation and improvement of experimental and computational proteomic strategies, such as separation techniques and the prediction of retention times and fragmentation patterns.

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, we report the development of a nanoPOTS (Nanodroplet Processing in One pot for Trace Samples) platform as a major advance in overall sensitivity. NanoPOTS dramatically enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive LC-MS, nanoPOTS allows identification of ~1500 to ~3,000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runsmore » algorithm of MaxQuant, >3000 proteins were consistently identified from as few as 10 cells. Furthermore, we demonstrate robust quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.« less

ZikaVR: An Integrated Zika Virus Resource for Genomics, Proteomics, Phylogenetic and Therapeutic Analysis

PubMed Central

Gupta, Amit Kumar; Kaur, Karambir; Rajput, Akanksha; Dhanda, Sandeep Kumar; Sehgal, Manika; Khan, Md. Shoaib; Monga, Isha; Dar, Showkat Ahmad; Singh, Sandeep; Nagpal, Gandharva; Usmani, Salman Sadullah; Thakur, Anamika; Kaur, Gazaldeep; Sharma, Shivangi; Bhardwaj, Aman; Qureshi, Abid; Raghava, Gajendra Pal Singh; Kumar, Manoj

2016-01-01

Current Zika virus (ZIKV) outbreaks that spread in several areas of Africa, Southeast Asia, and in pacific islands is declared as a global health emergency by World Health Organization (WHO). It causes Zika fever and illness ranging from severe autoimmune to neurological complications in humans. To facilitate research on this virus, we have developed an integrative multi-omics platform; ZikaVR (http://bioinfo.imtech.res.in/manojk/zikavr/), dedicated to the ZIKV genomic, proteomic and therapeutic knowledge. It comprises of whole genome sequences, their respective functional information regarding proteins, genes, and structural content. Additionally, it also delivers sophisticated analysis such as whole-genome alignments, conservation and variation, CpG islands, codon context, usage bias and phylogenetic inferences at whole genome and proteome level with user-friendly visual environment. Further, glycosylation sites and molecular diagnostic primers were also analyzed. Most importantly, we also proposed potential therapeutically imperative constituents namely vaccine epitopes, siRNAs, miRNAs, sgRNAs and repurposing drug candidates. PMID:27633273

Proteomic responses of fruits to environmental stresses

PubMed Central

Chan, Zhulong

2012-01-01

Fruits and vegetables are extremely susceptible to decay and easily lose commercial value after harvest. Different strategies have been developed to control postharvest decay and prevent quality deterioration during postharvest storage, including cold storage, controlled atmosphere (CA), and application of biotic and abiotic stimulus. In this review, mechanisms related to protein level responses of host side and pathogen side were characterized. Protein extraction protocols have been successfully developed for recalcitrant, low protein content fruit tissues. Comparative proteome profiling and functional analysis revealed that defense related proteins, energy metabolism, and antioxidant pathway played important roles in fruits in response to storage conditions and exogenous elicitor treatments. Secretome of pathogenic fungi has been well-investigated and the results indicated that hydrolytic enzymes were the key virulent factors for the pathogen infection. These protein level changes shed new light on interaction among fruits, pathogens, and environmental conditions. Potential postharvest strategies to reduce risk of fruit decay were further proposed based on currently available proteomic data. PMID:23335934

Proteomic evaluation of genetically modified crops: current status and challenges.

PubMed

Gong, Chun Yan; Wang, Tai

2013-01-01

Hectares of genetically modified (GM) crops have increased exponentially since 1996, when such crops began to be commercialized. GM biotechnology, together with conventional breeding, has become the main approach to improving agronomic traits of crops. However, people are concerned about the safety of GM crops, especially GM-derived food and feed. Many efforts have been made to evaluate the unintended effects caused by the introduction of exogenous genes. "Omics" techniques have advantages over targeted analysis in evaluating such crops because of their use of high-throughput screening. Proteins are key players in gene function and are directly involved in metabolism and cellular development or have roles as toxins, antinutrients, or allergens, which are essential for human health. Thus, proteomics can be expected to become one of the most useful tools in safety assessment. This review assesses the potential of proteomics in evaluating various GM crops. We further describe the challenges in ensuring homogeneity and sensitivity in detection techniques.

Plants versus Fungi and Oomycetes: Pathogenesis, Defense and Counter-Defense in the Proteomics Era

PubMed Central

El Hadrami, Abdelbasset; El-Bebany, Ahmed F.; Yao, Zhen; Adam, Lorne R.; El Hadrami, Ismailx; Daayf, Fouad

2012-01-01

Plant-fungi and plant-oomycete interactions have been studied at the proteomic level for many decades. However, it is only in the last few years, with the development of new approaches, combined with bioinformatics data mining tools, gel staining, and analytical instruments, such as 2D-PAGE/nanoflow-LC-MS/MS, that proteomic approaches thrived. They allow screening and analysis, at the sub-cellular level, of peptides and proteins resulting from plants, pathogens, and their interactions. They also highlight post-translational modifications to proteins, e.g., glycosylation, phosphorylation or cleavage. However, many challenges are encountered during in planta studies aimed at stressing details of host defenses and fungal and oomycete pathogenicity determinants during interactions. Dissecting the mechanisms of such host-pathogen systems, including pathogen counter-defenses, will ensure a step ahead towards understanding current outcomes of interactions from a co-evolutionary point of view, and eventually move a step forward in building more durable strategies for management of diseases caused by fungi and oomycetes. Unraveling intricacies of more complex proteomic interactions that involve additional microbes, i.e., PGPRs and symbiotic fungi, which strengthen plant defenses will generate valuable information on how pathosystems actually function in nature, and thereby provide clues to solving disease problems that engender major losses in crops every year. PMID:22837691

Simultaneous Proteomic Discovery and Targeted Monitoring using Liquid Chromatography, Ion Mobility Spectrometry, and Mass Spectrometry.

PubMed

Burnum-Johnson, Kristin E; Nie, Song; Casey, Cameron P; Monroe, Matthew E; Orton, Daniel J; Ibrahim, Yehia M; Gritsenko, Marina A; Clauss, Therese R W; Shukla, Anil K; Moore, Ronald J; Purvine, Samuel O; Shi, Tujin; Qian, Weijun; Liu, Tao; Baker, Erin S; Smith, Richard D

2016-12-01

Current proteomic approaches include both broad discovery measurements and quantitative targeted analyses. In many cases, discovery measurements are initially used to identify potentially important proteins (e.g. candidate biomarkers) and then targeted studies are employed to quantify a limited number of selected proteins. Both approaches, however, suffer from limitations. Discovery measurements aim to sample the whole proteome but have lower sensitivity, accuracy, and quantitation precision than targeted approaches, whereas targeted measurements are significantly more sensitive but only sample a limited portion of the proteome. Herein, we describe a new approach that performs both discovery and targeted monitoring (DTM) in a single analysis by combining liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled target peptides are spiked into tryptic digests and both the labeled and unlabeled peptides are detected using LC-IMS-MS instrumentation. Compared with the broad LC-MS discovery measurements, DTM yields greater peptide/protein coverage and detects lower abundance species. DTM also achieved detection limits similar to selected reaction monitoring (SRM) indicating its potential for combined high quality discovery and targeted analyses, which is a significant step toward the convergence of discovery and targeted approaches. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

PubMed

Dormeyer, Wilma; van Hoof, Dennis; Mummery, Christine L; Krijgsveld, Jeroen; Heck, Albert J R

2008-10-01

The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological mechanisms that regulate proliferation and differentiation. The comparison of their membrane proteomes will help unravel the biological principles of pluripotency, and the identification of biomarker proteins in their plasma membranes is considered a crucial step to fully exploit pluripotent cells for therapeutic purposes. For these tasks, membrane proteomics is the method of choice, but as indicated by the scarce identification of membrane and plasma membrane proteins in global proteomic surveys it is not an easy task. In this minireview, we first describe the general challenges of membrane proteomics. We then review current sample preparation steps and discuss protocols that we found particularly beneficial for the identification of large numbers of (plasma) membrane proteins in human tumour- and embryo-derived stem cells. Our optimized assembled protocol led to the identification of a large number of membrane proteins. However, as the composition of cells and membranes is highly variable we still recommend adapting the sample preparation protocol for each individual system.

Plants versus fungi and oomycetes: pathogenesis, defense and counter-defense in the proteomics era.

PubMed

El Hadrami, Abdelbasset; El-Bebany, Ahmed F; Yao, Zhen; Adam, Lorne R; El Hadrami, Ismailx; Daayf, Fouad

2012-01-01

Plant-fungi and plant-oomycete interactions have been studied at the proteomic level for many decades. However, it is only in the last few years, with the development of new approaches, combined with bioinformatics data mining tools, gel staining, and analytical instruments, such as 2D-PAGE/nanoflow-LC-MS/MS, that proteomic approaches thrived. They allow screening and analysis, at the sub-cellular level, of peptides and proteins resulting from plants, pathogens, and their interactions. They also highlight post-translational modifications to proteins, e.g., glycosylation, phosphorylation or cleavage. However, many challenges are encountered during in planta studies aimed at stressing details of host defenses and fungal and oomycete pathogenicity determinants during interactions. Dissecting the mechanisms of such host-pathogen systems, including pathogen counter-defenses, will ensure a step ahead towards understanding current outcomes of interactions from a co-evolutionary point of view, and eventually move a step forward in building more durable strategies for management of diseases caused by fungi and oomycetes. Unraveling intricacies of more complex proteomic interactions that involve additional microbes, i.e., PGPRs and symbiotic fungi, which strengthen plant defenses will generate valuable information on how pathosystems actually function in nature, and thereby provide clues to solving disease problems that engender major losses in crops every year.

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.

Meta-proteomic analysis of protein expression distinctive to electricity-generating biofilm communities in air-cathode microbial fuel cells.

PubMed

Chignell, Jeremy F; De Long, Susan K; Reardon, Kenneth F

2018-01-01

Bioelectrochemical systems (BESs) harness electrons from microbial respiration to generate power or chemical products from a variety of organic feedstocks, including lignocellulosic biomass, fermentation byproducts, and wastewater sludge. In some BESs, such as microbial fuel cells (MFCs), bacteria living in a biofilm use the anode as an electron acceptor for electrons harvested from organic materials such as lignocellulosic biomass or waste byproducts, generating energy that may be used by humans. Many BES applications use bacterial biofilm communities, but no studies have investigated protein expression by the anode biofilm community as a whole. To discover functional protein expression during current generation that may be useful for MFC optimization, a label-free meta-proteomics approach was used to compare protein expression in acetate-fed anode biofilms before and after the onset of robust electricity generation. Meta-proteomic comparisons were integrated with 16S rRNA gene-based community analysis at four developmental stages. The community composition shifted from dominance by aerobic Gammaproteobacteria (90.9 ± 3.3%) during initial biofilm formation to dominance by Deltaproteobacteria , particularly Geobacter (68.7 ± 3.6%) in mature, electricity-generating anodes. Community diversity in the intermediate stage, just after robust current generation began, was double that at the early stage and nearly double that of mature anode communities. Maximum current densities at the intermediate stage, however, were relatively similar (~ 83%) to those achieved by mature-stage biofilms. Meta-proteomic analysis, correlated with population changes, revealed significant enrichment of categories specific to membrane and transport functions among proteins from electricity-producing biofilms. Proteins detected only in electricity-producing biofilms were associated with gluconeogenesis, the glyoxylate cycle, and fatty acid β-oxidation, as well as with denitrification and competitive inhibition. The results demonstrate that it is possible for an MFC microbial community to generate robust current densities while exhibiting high taxonomic diversity. Moreover, these data provide evidence to suggest that startup growth of air-cathode MFCs under conditions that promote the establishment of aerobic-anaerobic syntrophy may decrease startup times. This study represents the first investigation into protein expression of a complex BES anode biofilm community as a whole. The findings contribute to understanding of the molecular mechanisms at work during BES startup and suggest options for improvement of BES generation of bioelectricity from renewable biomass.

Dissection of the Human Multipotent Adult Progenitor Cell Secretome by Proteomic Analysis

PubMed Central

van't Hof, Wouter; Newell, Laura F.; Reddy, Ashok; Wilmarth, Phillip A.; David, Larry L.; Raber, Amy; Bogaerts, Annelies; Pinxteren, Jef; Deans, Robert J.; Maziarz, Richard T.

2013-01-01

Multipotent adult progenitor cells (MAPCs) are adult adherent stromal stem cells currently being assessed in acute graft versus host disease clinical trials with demonstrated immunomodulatory capabilities and the potential to ameliorate detrimental autoimmune and inflammation-related processes. Our previous studies documented that MAPCs secrete factors that play a role in regulating T-cell activity. Here we expand our studies using a proteomics approach to characterize and quantify MAPC secretome components secreted over 72 hours in vitro under steady-state conditions and in the presence of the inflammatory triggers interferon-γ and lipopolysaccharide, or a tolerogenic CD74 ligand, RTL1000. MAPCs differentially responded to each of the tested stimuli, secreting molecules that regulate the biological activity of the extracellular matrix (ECM), including proteins that make up the ECM itself, proteins that regulate its construction/deconstruction, and proteins that serve to attach and detach growth factors from ECM components for redistribution upon appropriate stimulation. MAPCs secreted a wide array of proteases, some detectable in their zymogen forms. MAPCs also secreted protease inhibitors that would regulate protease activity. MAPCs secreted chemokines and cytokines that could provide molecular guidance cues to various cell types, including neutrophils, macrophages, and T cells. In addition, MAPCs secreted factors involved in maintenance of a homeostatic environment, regulating such diverse programs as innate immunity, angiogenesis/angiostasis, targeted delivery of growth factors, and the matrix-metalloprotease cascade. PMID:23981727

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

CONVERGENT TRANSCRIPTOMICS AND PROTEOMICS OF ENVIRONMENTAL ENRICHMENT AND COCAINE IDENTIFIES NOVEL THERAPEUTIC STRATEGIES FOR ADDICTION

PubMed Central

ZHANG, YAFANG; CROFTON, ELIZABETH J.; FAN, XIUZHEN; LI, DINGGE; KONG, FANPING; SINHA, MALA; LUXON, BRUCE A.; SPRATT, HEIDI M.; LICHTI, CHERYL F.; GREEN, THOMAS A.

2016-01-01

Transcriptomic and proteomic approaches have separately proven effective at identifying novel mechanisms affecting addiction-related behavior; however, it is difficult to prioritize the many promising leads from each approach. A convergent secondary analysis of proteomic and transcriptomic results can glean additional information to help prioritize promising leads. The current study is a secondary analysis of the convergence of recently published separate transcriptomic and proteomic analyses of nucleus accumbens (NAc) tissue from rats subjected to environmental enrichment vs. isolation and cocaine self-administration vs. saline. Multiple bioinformatics approaches (e.g. Gene Ontology (GO) analysis, Ingenuity Pathway Analysis (IPA), and Gene Set Enrichment Analysis (GSEA)) were used to interrogate these rich data sets. Although there was little correspondence between mRNA vs. protein at the individual target level, good correspondence was found at the level of gene/protein sets, particularly for the environmental enrichment manipulation. These data identify gene sets where there is a positive relationship between changes in mRNA and protein (e.g. glycolysis, ATP synthesis, translation elongation factor activity, etc.) and gene sets where there is an inverse relationship (e.g. ribosomes, Rho GTPase signaling, protein ubiquitination, etc.). Overall environmental enrichment produced better correspondence than cocaine self-administration. The individual targets contributing to mRNA and protein effects were largely not overlapping. As a whole, these results confirm that robust transcriptomic and proteomic data sets can provide similar results at the gene/protein set level even when there is little correspondence at the individual target level and little overlap in the targets contributing to the effects. PMID:27717806

Correlation of proteome-wide changes with social immunity behaviors provides insight into resistance to the parasitic mite, Varroa destructor, in the honey bee (Apis mellifera)

PubMed Central

2012-01-01

Background Disease is a major factor driving the evolution of many organisms. In honey bees, selection for social behavioral responses is the primary adaptive process facilitating disease resistance. One such process, hygienic behavior, enables bees to resist multiple diseases, including the damaging parasitic mite Varroa destructor. The genetic elements and biochemical factors that drive the expression of these adaptations are currently unknown. Proteomics provides a tool to identify proteins that control behavioral processes, and these proteins can be used as biomarkers to aid identification of disease tolerant colonies. Results We sampled a large cohort of commercial queen lineages, recording overall mite infestation, hygiene, and the specific hygienic response to V. destructor. We performed proteome-wide correlation analyses in larval integument and adult antennae, identifying several proteins highly predictive of behavior and reduced hive infestation. In the larva, response to wounding was identified as a key adaptive process leading to reduced infestation, and chitin biosynthesis and immune responses appear to represent important disease resistant adaptations. The speed of hygienic behavior may be underpinned by changes in the antenna proteome, and chemosensory and neurological processes could also provide specificity for detection of V. destructor in antennae. Conclusions Our results provide, for the first time, some insight into how complex behavioural adaptations manifest in the proteome of honey bees. The most important biochemical correlations provide clues as to the underlying molecular mechanisms of social and innate immunity of honey bees. Such changes are indicative of potential divergence in processes controlling the hive-worker maturation. PMID:23021491

Correlation of proteome-wide changes with social immunity behaviors provides insight into resistance to the parasitic mite, Varroa destructor, in the honey bee (Apis mellifera).

PubMed

Parker, Robert; Guarna, M Marta; Melathopoulos, Andony P; Moon, Kyung-Mee; White, Rick; Huxter, Elizabeth; Pernal, Stephen F; Foster, Leonard J

2012-06-29

Disease is a major factor driving the evolution of many organisms. In honey bees, selection for social behavioral responses is the primary adaptive process facilitating disease resistance. One such process, hygienic behavior, enables bees to resist multiple diseases, including the damaging parasitic mite Varroa destructor. The genetic elements and biochemical factors that drive the expression of these adaptations are currently unknown. Proteomics provides a tool to identify proteins that control behavioral processes, and these proteins can be used as biomarkers to aid identification of disease tolerant colonies. We sampled a large cohort of commercial queen lineages, recording overall mite infestation, hygiene, and the specific hygienic response to V. destructor. We performed proteome-wide correlation analyses in larval integument and adult antennae, identifying several proteins highly predictive of behavior and reduced hive infestation. In the larva, response to wounding was identified as a key adaptive process leading to reduced infestation, and chitin biosynthesis and immune responses appear to represent important disease resistant adaptations. The speed of hygienic behavior may be underpinned by changes in the antenna proteome, and chemosensory and neurological processes could also provide specificity for detection of V. destructor in antennae. Our results provide, for the first time, some insight into how complex behavioural adaptations manifest in the proteome of honey bees. The most important biochemical correlations provide clues as to the underlying molecular mechanisms of social and innate immunity of honey bees. Such changes are indicative of potential divergence in processes controlling the hive-worker maturation.

Redox regulation of mitochondrial proteins and proteomes by cysteine thiol switches.

PubMed

Nietzel, Thomas; Mostertz, Jörg; Hochgräfe, Falko; Schwarzländer, Markus

2017-03-01

Mitochondria are hotspots of cellular redox biochemistry. Respiration as a defining mitochondrial function is made up of a series of electron transfers that are ultimately coupled to maintaining the proton motive force, ATP production and cellular energy supply. The individual reaction steps involved require tight control and flexible regulation to maintain energy and redox balance in the cell under fluctuating demands. Redox regulation by thiol switching has been a long-standing candidate mechanism to support rapid adjustment of mitochondrial protein function at the posttranslational level. Here we review recent advances in our understanding of cysteine thiol switches in the mitochondrial proteome with a focus on their operation in vivo. We assess the conceptual basis for thiol switching in mitochondria and discuss to what extent insights gained from in vitro studies may be valid in vivo, considering thermodynamic, kinetic and structural constraints. We compare functional proteomic approaches that have been used to assess mitochondrial protein thiol switches, including thioredoxin trapping, redox difference gel electrophoresis (redoxDIGE), isotope-coded affinity tag (OxICAT) and iodoacetyl tandem mass tag (iodoTMT) labelling strategies. We discuss conditions that may favour active thiol switching in mitochondrial proteomes in vivo, and appraise recent advances in dissecting their impact using combinations of in vivo redox sensing and quantitative redox proteomics. Finally we focus on four central facets of mitochondrial biology, aging, carbon metabolism, energy coupling and electron transport, exemplifying the current emergence of a mechanistic understanding of mitochondrial regulation by thiol switching in living plants and animals. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

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

A Combined Metabonomic and Proteomic Approach Identifies Frontal Cortex Changes in a Chronic Phencyclidine Rat Model in Relation to Human Schizophrenia Brain Pathology

PubMed Central

Wesseling, Hendrik; Chan, Man K; Tsang, T M; Ernst, Agnes; Peters, Fabian; Guest, Paul C; Holmes, Elaine; Bahn, Sabine

2013-01-01

Current schizophrenia (SCZ) treatments fail to treat the broad range of manifestations associated with this devastating disorder. Thus, new translational models that reproduce the core pathological features are urgently needed to facilitate novel drug discovery efforts. Here, we report findings from the first comprehensive label-free liquid-mass spectrometry proteomic- and proton nuclear magnetic resonance-based metabonomic profiling of the rat frontal cortex after chronic phencyclidine (PCP) intervention, which induces SCZ-like symptoms. The findings were compared with results from a proteomic profiling of post-mortem prefrontal cortex from SCZ patients and with relevant findings in the literature. Through this approach, we identified proteomic alterations in glutamate-mediated Ca2+ signaling (Ca2+/calmodulin-dependent protein kinase II, PPP3CA, and VISL1), mitochondrial function (GOT2 and PKLR), and cytoskeletal remodeling (ARP3). Metabonomic profiling revealed changes in the levels of glutamate, glutamine, glycine, pyruvate, and the Ca2+ regulator taurine. Effects on similar pathways were also identified in the prefrontal cortex tissue from human SCZ subjects. The discovery of similar but not identical proteomic and metabonomic alterations in the chronic PCP rat model and human brain indicates that this model recapitulates only some of the molecular alterations of the disease. This knowledge may be helpful in understanding mechanisms underlying psychosis, which, in turn, can facilitate improved therapy and drug discovery for SCZ and other psychiatric diseases. Most importantly, these molecular findings suggest that the combined use of multiple models may be required for more effective translation to studies of human SCZ. PMID:23942359

Plasma-based proteomics reveals immune response, complement and coagulation cascades pathway shifts in heat-stressed lactating dairy cows.

PubMed

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

2016-09-02

Heat stress (HS) has an enormous economic impact on the dairy industry. In recent years, many researchers have investigated changes in the gene expression and metabolomics profiles in dairy cows caused by HS. However, the proteomics profiles of heat-stressed dairy cows have not yet been completely elucidated. We compared plasma proteomics from HS-free and heat-stressed dairy cows using an iTRAQ labeling approach. After the depletion of high abundant proteins in the plasma, 1472 proteins were identified. Of these, 85 proteins were differentially abundant in cows exposed to HS relative to HS-free. Database searches combined with GO and KEGG pathway enrichment analyses revealed that many components of the complement and coagulation cascades were altered in heat-stressed cows compared with HS-free cows. Of these, many factors in the complement system (including complement components C1, C3, C5, C6, C7, C8, and C9, complement factor B, and factor H) were down-regulated by HS, while components of the coagulation system (including coagulation factors, vitamin K-dependent proteins, and fibrinogens) were up-regulated by HS. In conclusion, our results indicate that HS decreases plasma levels of complement system proteins, suggesting that immune function is impaired in dairy cows exposed to HS. Though many aspects of heat stress (HS) have been extensively researched, relatively little is known about the proteomics profile changes that occur during heat exposure. In this work, we employed a proteomics approach to investigate differential abundance of plasma proteins in HS-free and heat-stressed dairy cows. Database searches combined with GO and KEGG pathway enrichment analyses revealed that HS resulted in a decrease in complement components, suggesting that heat-stressed dairy cows have impaired immune function. In addition, through integrative analyses of proteomics and previous metabolomics, we showed enhanced glycolysis, lipid metabolic pathway shifts, and nitrogen repartitioning in dairy cows exposed to HS. Our findings expand our current knowledge on the effects of HS on plasma proteomics in dairy cows and offer a new perspective for future research. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of the functional aspects and seminal plasma proteomic profile of sperm from smokers.

PubMed

Antoniassi, Mariana Pereira; Intasqui, Paula; Camargo, Mariana; Zylbersztejn, Daniel Suslik; Carvalho, Valdemir Melechco; Cardozo, Karina H M; Bertolla, Ricardo Pimenta

2016-11-01

To evaluate the effect of smoking on sperm functional quality and seminal plasma proteomic profile. Sperm functional tests were performed in 20 non-smoking men with normal semen quality, according to the World Health Organization (2010) and in 20 smoking patients. These included: evaluation of DNA fragmentation by alkaline Comet assay; analysis of mitochondrial activity using DAB staining; and acrosomal integrity evaluation by PNA binding. The remaining semen was centrifuged and seminal plasma was used for proteomic analysis (liquid chromatography-tandem mass spectrometry). The quantified proteins were used for Venn diagram construction in Cytoscape 3.2.1 software, using the PINA4MS plug-in. Then, differentially expressed proteins were used for functional enrichment analysis of Gene Ontology categories, Kyoto Encyclopedia of Genes and Genomes and Reactome, using Cytoscape software and the ClueGO 2.2.0 plug-in. Smokers had a higher percentage of sperm DNA damage (Comet classes III and IV; P < 0.01), partially and fully inactive mitochondria (DAB classes III and IV; P = 0.001 and P = 0.006, respectively) and non-intact acrosomes (P < 0.01) when compared with the control group. With respect to proteomic analysis, 422 proteins were identified and quantified, of which one protein was absent, 27 proteins were under-represented and six proteins were over-represented in smokers. Functional enrichment analysis showed the enrichment of antigen processing and presentation, positive regulation of prostaglandin secretion involved in immune response, protein kinase A signalling and arachidonic acid secretion, complement activation, regulation of the cytokine-mediated signalling pathway and regulation of acute inflammatory response in the study group (smokers). In conclusion, cigarette smoking was associated with an inflammatory state in the accessory glands and in the testis, as shown by enriched proteomic pathways. This state causes an alteration in sperm functional quality, which is characterized by decreased acrosome integrity and mitochondrial activity, as well as by increased nuclear DNA fragmentation. © 2016 The Authors BJU International © 2016 BJU International Published by John Wiley & Sons Ltd.

The quantitative and condition-dependent Escherichia coli proteome

PubMed Central

Schmidt, Alexander; Kochanowski, Karl; Vedelaar, Silke; Ahrné, Erik; Volkmer, Benjamin; Callipo, Luciano; Knoops, Kèvin; Bauer, Manuel; Aebersold, Ruedi; Heinemann, Matthias

2016-01-01

Measuring precise concentrations of proteins can provide insights into biological processes. Here, we use efficient protein extraction and sample fractionation and state-of-the-art quantitative mass spectrometry techniques to generate a comprehensive, condition-dependent protein abundance map of Escherichia coli. We measure cellular protein concentrations for 55% of predicted E. coli genes (>2300 proteins) under 22 different experimental conditions and identify methylation and N-terminal protein acetylations previously not known to be prevalent in bacteria. We uncover system-wide proteome allocation, expression regulation, and post-translational adaptations. These data provide a valuable resource for the systems biology and broader E. coli research communities. PMID:26641532

PROTEOMIC ANALYSIS OF ALLERGENS FROM METARHIZIUM ANISOPLIAE

EPA Science Inventory

Introduction

The goal of this project is the identification and characterization of allergens from the fungus Metarhizium anisopliae, using mass spectrometry (MS). The US EPA, under the "Children at Risk" program, is currently addressing the problem of indoor fungal bioaer...

PROTEOMIC ANALYSIS OF ALLERGENS FROM METARHIZIUM ANISOPLIEA

EPA Science Inventory

The goal of this project is the identification and characterization of allergens from the fungus M. Anisopliae, using mass spectrometry (MS). The US EPA, under the "Children at Risk" program, is currently addressing the problem of indoor fungal bioaerosol contamination. One of ...

Corra: Computational framework and tools for LC-MS discovery and targeted mass spectrometry-based proteomics

PubMed Central

Brusniak, Mi-Youn; Bodenmiller, Bernd; Campbell, David; Cooke, Kelly; Eddes, James; Garbutt, Andrew; Lau, Hollis; Letarte, Simon; Mueller, Lukas N; Sharma, Vagisha; Vitek, Olga; Zhang, Ning; Aebersold, Ruedi; Watts, Julian D

2008-01-01

Background Quantitative proteomics holds great promise for identifying proteins that are differentially abundant between populations representing different physiological or disease states. A range of computational tools is now available for both isotopically labeled and label-free liquid chromatography mass spectrometry (LC-MS) based quantitative proteomics. However, they are generally not comparable to each other in terms of functionality, user interfaces, information input/output, and do not readily facilitate appropriate statistical data analysis. These limitations, along with the array of choices, present a daunting prospect for biologists, and other researchers not trained in bioinformatics, who wish to use LC-MS-based quantitative proteomics. Results We have developed Corra, a computational framework and tools for discovery-based LC-MS proteomics. Corra extends and adapts existing algorithms used for LC-MS-based proteomics, and statistical algorithms, originally developed for microarray data analyses, appropriate for LC-MS data analysis. Corra also adapts software engineering technologies (e.g. Google Web Toolkit, distributed processing) so that computationally intense data processing and statistical analyses can run on a remote server, while the user controls and manages the process from their own computer via a simple web interface. Corra also allows the user to output significantly differentially abundant LC-MS-detected peptide features in a form compatible with subsequent sequence identification via tandem mass spectrometry (MS/MS). We present two case studies to illustrate the application of Corra to commonly performed LC-MS-based biological workflows: a pilot biomarker discovery study of glycoproteins isolated from human plasma samples relevant to type 2 diabetes, and a study in yeast to identify in vivo targets of the protein kinase Ark1 via phosphopeptide profiling. Conclusion The Corra computational framework leverages computational innovation to enable biologists or other researchers to process, analyze and visualize LC-MS data with what would otherwise be a complex and not user-friendly suite of tools. Corra enables appropriate statistical analyses, with controlled false-discovery rates, ultimately to inform subsequent targeted identification of differentially abundant peptides by MS/MS. For the user not trained in bioinformatics, Corra represents a complete, customizable, free and open source computational platform enabling LC-MS-based proteomic workflows, and as such, addresses an unmet need in the LC-MS proteomics field. PMID:19087345

The Proteomics Stock Market Project. A Cross-Disciplinary Collaboration in Biochemistry and Business Education

NASA Astrophysics Data System (ADS)

Keller, Heath; Cox, James R.

2004-04-01

Students taking courses in different disciplines can work together to add unique elements to their educational experience. A model for this type of pedagogical approach has been established in the Proteomics Stock Market Project, a collaborative effort between instructors and students in the Department of Chemistry and Department of Management, Marketing, and Business Administration at Murray State University. Stage I involved biochemistry students investigating the topic of proteomics and choosing companies for potential investment based only on scientific investigation. Marketing and management students completed Stage II and provided an investment analysis on the companies selected in Stage I. In Stage III, the biochemistry students focused on a particular company and investigated a protein-based therapeutic product. Blackboard software was utilized in each stage of the project to facilitate the exchange of information and electronic documents. This project was designed to give biochemistry students an appreciation for the emerging field of proteomics and the marketing and management students a flavor for real-world applications of business principles. During the project, students were exposed to ideas and concepts not typically covered in their courses. With this involvement, the students had the opportunity to gain a broader perspective of course content compared to a more traditional curriculum.

Proteomic Analysis of Lyme Disease: Global Protein Comparison of Three Strains of Borrelia burgdorferi

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

Jacobs, Jon M.; Yang, Xiaohua; Luft, Benjamin J.

2005-04-01

The Borrelia burgdorferi spirochete is the causative agent of Lyme disease, the most common tick-borne disease in the United States. It has been studied extensively to help understand its pathogenicity of infection and how it can persist in different mammalian hosts. We report the proteomic analysis of the archetype B. burgdorferi B31 strain and two other strains (ND40, and JD-1) having different Borrelia pathotypes using strong cation exchange fractionation of proteolytic peptides followed by high-resolution, reversed phase capillary liquid chromatography coupled with ion trap tandem mass spectrometric (LC-MS/MS) analysis. Protein identification was facilitated by the availability of the complete B31more » genome sequence. A total of 665 Borrelia proteins were identified representing ~38 % coverage of the theoretical B31 proteome. A significant overlap was observed between the identified proteins in direct comparisons between any two strains (>72%), but distinct differences were observed among identified hypothetical and outer membrane proteins of the three strains. Such a concurrent proteomic overview of three Borrelia strains based upon only the B31 genome sequence is shown to provide significant insights into the presence or absence of specific proteins and a broad overall comparison among strains.« less

iTRAQ-Based Quantitative Proteomics of Developing and Ripening Muscadine Grape Berry

PubMed Central

Kambiranda, Devaiah; Katam, Ramesh; Basha, Sheikh M.; Siebert, Shalom

2014-01-01

Grapes are among the widely cultivated fruit crops in the world. Grape berries like other nonclimacteric fruits undergo a complex set of dynamic, physical, physiological, and biochemical changes during ripening. Muscadine grapes are widely cultivated in the southern United States for fresh fruit and wine. To date, changes in the metabolites composition of muscadine grapes have been well documented; however, the molecular changes during berry development and ripening are not fully known. The aim of this study was to investigate changes in the berry proteome during ripening in muscadine grape cv. Noble. Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS was used to detect statistically significant changes in the berry proteome. A total of 674 proteins were detected, and 76 were differentially expressed across four time points in muscadine berry. Proteins obtained were further analyzed to provide information about its potential functions during ripening. Several proteins involved in abiotic and biotic stimuli and sucrose and hexose metabolism were upregulated during berry ripening. Quantitative real-time PCR analysis validated the protein expression results for nine proteins. Identification of vicilin-like antimicrobial peptides indicates additional disease tolerance proteins are present in muscadines for berry protection during ripening. The results provide new information for characterization and understanding muscadine berry proteome and grape ripening. PMID:24251720

A standardized framing for reporting protein identifications in mzIdentML 1.2

PubMed Central

Seymour, Sean L.; Farrah, Terry; Binz, Pierre-Alain; Chalkley, Robert J.; Cottrell, John S.; Searle, Brian C.; Tabb, David L.; Vizcaíno, Juan Antonio; Prieto, Gorka; Uszkoreit, Julian; Eisenacher, Martin; Martínez-Bartolomé, Salvador; Ghali, Fawaz; Jones, Andrew R.

2015-01-01

Inferring which protein species have been detected in bottom-up proteomics experiments has been a challenging problem for which solutions have been maturing over the past decade. While many inference approaches now function well in isolation, comparing and reconciling the results generated across different tools remains difficult. It presently stands as one of the greatest barriers in collaborative efforts such as the Human Proteome Project and public repositories like the PRoteomics IDEntifications (PRIDE) database. Here we present a framework for reporting protein identifications that seeks to improve capabilities for comparing results generated by different inference tools. This framework standardizes the terminology for describing protein identification results, associated with the HUPO-Proteomics Standards Initiative (PSI) mzIdentML standard, while still allowing for differing methodologies to reach that final state. It is proposed that developers of software for reporting identification results will adopt this terminology in their outputs. While the new terminology does not require any changes to the core mzIdentML model, it represents a significant change in practice, and, as such, the rules will be released via a new version of the mzIdentML specification (version 1.2) so that consumers of files are able to determine whether the new guidelines have been adopted by export software. PMID:25092112

Subfractionation, characterization and in-depth proteomic analysis of glomerular membrane vesicles in human urine

PubMed Central

Hogan, Marie C.; Johnson, Kenneth L.; Zenka, Roman M.; Charlesworth, M. Cristine; Madden, Benjamin J.; Mahoney, Doug W.; Oberg, Ann L.; Huang, Bing Q.; Nesbitt, Lisa L.; Bakeberg, Jason L.; Bergen, H. Robert; Ward, Christopher J.

2014-01-01

Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40–200nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV) enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin positive irregularly shaped membranous vesicles and podocin/podocalyxin negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton and RhoGDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases and complement proteins involved in glomerular disease are in GMVs and some were shed in the disease state (nephrin, TRPC6 and INF2 and PLA2R). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease. PMID:24196483

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.

Functional insights from proteome-wide structural modeling of Treponema pallidum subspecies pallidum, the causative agent of syphilis.

PubMed

Houston, Simon; Lithgow, Karen Vivien; Osbak, Kara Krista; Kenyon, Chris Richard; Cameron, Caroline E

2018-05-16

Syphilis continues to be a major global health threat with 11 million new infections each year, and a global burden of 36 million cases. The causative agent of syphilis, Treponema pallidum subspecies pallidum, is a highly virulent bacterium, however the molecular mechanisms underlying T. pallidum pathogenesis remain to be definitively identified. This is due to the fact that T. pallidum is currently uncultivatable, inherently fragile and thus difficult to work with, and phylogenetically distinct with no conventional virulence factor homologs found in other pathogens. In fact, approximately 30% of its predicted protein-coding genes have no known orthologs or assigned functions. Here we employed a structural bioinformatics approach using Phyre2-based tertiary structure modeling to improve our understanding of T. pallidum protein function on a proteome-wide scale. Phyre2-based tertiary structure modeling generated high-confidence predictions for 80% of the T. pallidum proteome (780/978 predicted proteins). Tertiary structure modeling also inferred the same function as primary structure-based annotations from genome sequencing pipelines for 525/605 proteins (87%), which represents 54% (525/978) of all T. pallidum proteins. Of the 175 T. pallidum proteins modeled with high confidence that were not assigned functions in the previously annotated published proteome, 167 (95%) were able to be assigned predicted functions. Twenty-one of the 175 hypothetical proteins modeled with high confidence were also predicted to exhibit significant structural similarity with proteins experimentally confirmed to be required for virulence in other pathogens. Phyre2-based structural modeling is a powerful bioinformatics tool that has provided insight into the potential structure and function of the majority of T. pallidum proteins and helped validate the primary structure-based annotation of more than 50% of all T. pallidum proteins with high confidence. This work represents the first T. pallidum proteome-wide structural modeling study and is one of few studies to apply this approach for the functional annotation of a whole proteome.

Targeted Data Extraction of the MS/MS Spectra Generated by Data-independent Acquisition: A New Concept for Consistent and Accurate Proteome Analysis*

PubMed Central

Gillet, Ludovic C.; Navarro, Pedro; Tate, Stephen; Röst, Hannes; Selevsek, Nathalie; Reiter, Lukas; Bonner, Ron; Aebersold, Ruedi

2012-01-01

Most proteomic studies use liquid chromatography coupled to tandem mass spectrometry to identify and quantify the peptides generated by the proteolysis of a biological sample. However, with the current methods it remains challenging to rapidly, consistently, reproducibly, accurately, and sensitively detect and quantify large fractions of proteomes across multiple samples. Here we present a new strategy that systematically queries sample sets for the presence and quantity of essentially any protein of interest. It consists of using the information available in fragment ion spectral libraries to mine the complete fragment ion maps generated using a data-independent acquisition method. For this study, the data were acquired on a fast, high resolution quadrupole-quadrupole time-of-flight (TOF) instrument by repeatedly cycling through 32 consecutive 25-Da precursor isolation windows (swaths). This SWATH MS acquisition setup generates, in a single sample injection, time-resolved fragment ion spectra for all the analytes detectable within the 400–1200 m/z precursor range and the user-defined retention time window. We show that suitable combinations of fragment ions extracted from these data sets are sufficiently specific to confidently identify query peptides over a dynamic range of 4 orders of magnitude, even if the precursors of the queried peptides are not detectable in the survey scans. We also show that queried peptides are quantified with a consistency and accuracy comparable with that of selected reaction monitoring, the gold standard proteomic quantification method. Moreover, targeted data extraction enables ad libitum quantification refinement and dynamic extension of protein probing by iterative re-mining of the once-and-forever acquired data sets. This combination of unbiased, broad range precursor ion fragmentation and targeted data extraction alleviates most constraints of present proteomic methods and should be equally applicable to the comprehensive analysis of other classes of analytes, beyond proteomics. PMID:22261725

Protein Equalizer Technology : the quest for a "democratic proteome".

PubMed

Righetti, Pier Giorgio; Boschetti, Egisto; Lomas, Lee; Citterio, Attilio

2006-07-01

No proteome can be considered "democratic", but rather "oligarchic", since a few proteins dominate the landscape and often obliterate the signal of the rare ones. This is the reason why most scientists lament that, in proteome analysis, the same set of abundant proteins is seen again and again. A host of pre-fractionation techniques have been described, but all of them, one way or another, are besieged by problems, in that they are based on a "depletion principle", i.e. getting rid of the unwanted species. Yet "democracy" calls not for killing the enemy, but for giving "equal rights" to all people. One way to achieve that would be the use of "Protein Equalizer Technology" for reducing protein concentration differences. This comprises a diverse library of combinatorial ligands coupled to spherical porous beads. When these beads come into contact with complex proteomes (e.g. human urine and serum, egg white, and any cell lysate, for that matter) of widely differing protein composition and relative abundances, they are able to "equalize" the protein population, by sharply reducing the concentration of the most abundant components, while simultaneously enhancing the concentration of the most dilute species. It is felt that this novel method could offer a strong step forward in bringing the "unseen proteome" (due to either low abundance and/or presence of interference) within the detection capabilities of current proteomics detection methods. Examples are given of equalization of human urine and serum samples, resulting in the discovery of a host of proteins never reported before. Additionally, these beads can be used to remove host cell proteins from purified recombinant proteins or protein purified from natural sources that are intended for human consumption. These proteins typically reach purities of the order of 98%: higher purities often become prohibitively expensive. Yet, if incubated with "equalizer beads", these last impurities can be effectively removed at a small cost and with minute losses of the main, valuable product.

Characterization of the canine urinary proteome.

PubMed

Brandt, Laura E; Ehrhart, E J; Scherman, Hataichanok; Olver, Christine S; Bohn, Andrea A; Prenni, Jessica E

2014-06-01

Urine is an attractive biofluid for biomarker discovery as it is easy and minimally invasive to obtain. While numerous studies have focused on the characterization of human urine, much less research has focused on canine urine. The objectives of this study were to characterize the universal canine urinary proteome (both soluble and exosomal), to determine the overlap between the canine proteome and a representative human urinary proteome study, to generate a resource for future canine studies, and to determine the suitability of the dog as a large animal model for human diseases. The soluble and exosomal fractions of normal canine urine were characterized using liquid chromatography tandem mass spectrometry (LC-MS/MS). Biological Networks Gene Ontology (BiNGO) software was utilized to assign the canine urinary proteome to respective Gene Ontology categories, such as Cellular Component, Molecular Function, and Biological Process. Over 500 proteins were confidently identified in normal canine urine. Gene Ontology analysis revealed that exosomal proteins were largely derived from an intracellular location, while soluble proteins included both extracellular and membrane proteins. Exosome proteins were assigned to metabolic processes and localization, while soluble proteins were primarily annotated to specific localization processes. Several proteins identified in normal canine urine have previously been identified in human urine where these proteins are related to various extrarenal and renal diseases. The results of this study illustrate the potential of the dog as an animal model for human disease states and provide the framework for future studies of canine renal diseases. © 2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology.

Systemic effects of ionizing radiation at the proteome and metabolome levels in the blood of cancer patients treated with radiotherapy: the influence of inflammation and radiation toxicity.

PubMed

Jelonek, Karol; Pietrowska, Monika; Widlak, Piotr

2017-07-01

Blood is the most common replacement tissue used to study systemic responses of organisms to different types of pathological conditions and environmental insults. Local irradiation during cancer radiotherapy induces whole body responses that can be observed at the blood proteome and metabolome levels. Hence, comparative blood proteomics and metabolomics are emerging approaches used in the discovery of radiation biomarkers. These techniques enable the simultaneous measurement of hundreds of molecules and the identification of sets of components that can discriminate different physiological states of the human body. Radiation-induced changes are affected by the dose and volume of irradiated tissues; hence, the molecular composition of blood is a hypothetical source of biomarkers for dose assessment and the prediction and monitoring of systemic responses to radiation. This review aims to provide a comprehensive overview on the available evidence regarding molecular responses to ionizing radiation detected at the level of the human blood proteome and metabolome. It focuses on patients exposed to radiation during cancer radiotherapy and emphasizes effects related to radiation-induced toxicity and inflammation. Systemic responses to radiation detected at the blood proteome and metabolome levels are primarily related to the intensity of radiation-induced toxicity, including inflammatory responses. Thus, several inflammation-associated molecules can be used to monitor or even predict radiation-induced toxicity. However, these abundant molecular features have a rather limited applicability as universal biomarkers for dose assessment, reflecting the individual predisposition of the immune system and tissue-specific mechanisms involved in radiation-induced damage.

Intrinsic circannual regulation of brown adipose tissue form and function in tune with hibernation

PubMed Central

Hindle, Allyson G.

2013-01-01

Winter hibernators repeatedly cycle between cold torpor and rewarming supported by nonshivering thermogenesis in brown adipose tissue (BAT). In contrast, summer animals are homeotherms, undergoing reproduction, growth, and fattening. This life history confers variability to BAT recruitment and activity. To address the components underlying prewinter enhancement and winter activation, we interrogated the BAT proteome in 13-lined ground squirrels among three summer and five winter states. We also examined mixed physiology in fall and spring individuals to test for ambient temperature and seasonal effects, as well as the timing of seasonal transitions. BAT form and function differ circannually in these animals, as evidenced by morphology and proteome dynamics. This intrinsic pattern distinguished homeothermic groups and early vs. late winter hibernators. Homeothermic variation derived from postemergence delay in growth and substrate biosynthesis. The heterothermic proteome varied less despite extreme winter physiological shifts and was optimized to exploit lipids by enhanced fatty acid binding, β-oxidation, and mitochondrial protein translocation. Surprisingly, ambient temperature did not affect the BAT proteome during transition seasons; rather, the pronounced summer-winter shift preceded environmental changes and phenotypic progression. During fall transition, differential regulation of two fatty acid binding proteins provides further evidence of recruitment and separates proteomic preparation from successful hibernation. Abundance of FABP4 correlates with torpor bout length throughout the year, clarifying its potential function in hibernation. Metabolically active BAT is a target for treating human obesity and metabolic disorders. Understanding the hibernator's extreme and seasonally distinct recruitment and activation control strategies offers untapped potential to identify novel, therapeutically relevant regulatory pathways. PMID:24326419

Comprehensive Analysis of Proteomic Differences between Escherichia coli K-12 and B Strains Using Multiplexed Isobaric Tandem Mass Tag (TMT) Labeling.

PubMed

Han, Mee-Jung

2017-11-28

The Escherichia coli K-12 and B strains are among the most frequently used bacterial hosts for scientific research and biotechnological applications. However, omics analyses have revealed that E. coli K-12 and B exhibit notably different genotypic and phenotypic attributes, even though they were derived from the same ancestor. In a previous study, we identified a limited number of proteins from the two strains using two-dimensional gel electrophoresis and tandem mass spectrometry (MS/MS). In this study, an in-depth analysis of the physiological behavior of the E. coli K-12 and B strains at the proteomic level was performed using six-plex isobaric tandem mass tag-based quantitative MS. Additionally, the best lysis buffer for increasing the efficiency of protein extraction was selected from three tested buffers prior to the quantitative proteomic analysis. This study identifies the largest number of proteins in the two E. coli strains reported to date and is the first to show the dynamics of these proteins. Notable differences in proteins associated with key cellular properties, including some metabolic pathways, the biosynthesis and degradation of amino acids, membrane integrity, cellular tolerance, and motility, were found between the two representative strains. Compared with previous studies, these proteomic results provide a more holistic view of the overall state of E. coli cells based on a single proteomic study and reveal significant insights into why the two strains show distinct phenotypes. Additionally, the resulting data provide in-depth information that will help fine-tune processes in the future.

Mining the human urine proteome for monitoring renal transplant injury

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

Sigdel, Tara K.; Gao, Yuqian; He, Jintang

The human urinary proteome reflects systemic and inherent renal injury perturbations and can be analyzed to harness specific biomarkers for different kidney transplant injury states. 396 unique urine samples were collected contemporaneously with an allograft biopsy from 396 unique kidney transplant recipients. Centralized, blinded histology on the graft was used to classify matched urine samples into categories of acute rejection (AR), chronic allograft nephropathy (CAN), BK virus nephritis (BKVN), and stable graft (STA). Liquid chromatography–mass spectrometry (LC-MS) based proteomics using iTRAQ based discovery (n=108) and global label-free LC-MS analyses of individual samples (n=137) for quantitative proteome assessment were used inmore » the discovery step. Selected reaction monitoring (SRM) was applied to identify and validate minimal urine protein/peptide biomarkers to accurately segregate organ injury causation and pathology on unique urine samples (n=151). A total of 958 proteins were initially quantified by iTRAQ, 87% of which were also identified among 1574 urine proteins detected in LC-MS validation. 103 urine proteins were significantly (p<0.05) perturbed in injury and enriched for humoral immunity, complement activation, and lymphocyte trafficking. A set of 131 peptides corresponding to 78 proteins were assessed by SRM for their significance in an independent sample cohort. A minimal set of 35 peptides mapping to 33 proteins, were modeled to segregate different injury groups (AUC =93% for AR, 99% for CAN, 83% for BKVN). Urinary proteome discovery and targeted validation identified urine protein fingerprints for non-invasive differentiation of kidney transplant injuries, thus opening the door for personalized immune risk assessment and therapy.« less

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

Gulf War Illness Inflammation Reduction Trial

DTIC Science & Technology

2017-10-01

United States military personnel were deployed to the Kuwaiti Theater of Operations during Operation Desert Shield and Operation Desert Storm (Gulf...differential, plasma proteomics, platelet function studies, and the measurement of multiple coagulation parameters. The pilot study results provide strong

Environmental proteomics reveals taxonomic and functional changes in an enriched aquatic ecosystem.

PubMed

Northrop, Amanda C; Brooks, Rachel; Ellison, Aaron M; Gotelli, Nicholas J; Ballif, Bryan A

2017-10-01

Aquatic ecosystem enrichment can lead to distinct and irreversible changes to undesirable states. Understanding changes in active microbial community function and composition following organic-matter loading in enriched ecosystems can help identify biomarkers of such state changes. In a field experiment, we enriched replicate aquatic ecosystems in the pitchers of the northern pitcher plant, Sarracenia purpurea . Shotgun metaproteomics using a custom metagenomic database identified proteins, molecular pathways, and contributing microbial taxa that differentiated control ecosystems from those that were enriched. The number of microbial taxa contributing to protein expression was comparable between treatments; however, taxonomic evenness was higher in controls. Functionally active bacterial composition differed significantly among treatments and was more divergent in control pitchers than enriched pitchers. Aerobic and facultative anaerobic bacteria contributed most to identified proteins in control and enriched ecosystems, respectively. The molecular pathways and contributing taxa in enriched pitcher ecosystems were similar to those found in larger enriched aquatic ecosystems and are consistent with microbial processes occurring at the base of detrital food webs. Detectable differences between protein profiles of enriched and control ecosystems suggest that a time series of environmental proteomics data may identify protein biomarkers of impending state changes to enriched states.

Proteoglycomics: Recent Progress and Future Challenges

PubMed Central

Ly, Mellisa; Laremore, Tatiana N.

2010-01-01

Abstract Proteoglycomics is a systematic study of structure, expression, and function of proteoglycans, a posttranslationally modified subset of a proteome. Although relying on the established technologies of proteomics and glycomics, proteoglycomics research requires unique approaches for elucidating structure–function relationships of both proteoglycan components, glycosaminoglycan chain, and core protein. This review discusses our current understanding of structure and function of proteoglycans, major players in the development, normal physiology, and disease. A brief outline of the proteoglycomic sample preparation and analysis is provided along with examples of several recent proteoglycomic studies. Unique challenges in the characterization of glycosaminoglycan component of proteoglycans are discussed, with emphasis on the many analytical tools used and the types of information they provide. PMID:20450439

Protein biomarkers of alcohol abuse

PubMed Central

Torrente, Mariana P; Freeman, Willard M; Vrana, Kent E

2012-01-01

Alcohol abuse can lead to a number of health and social issues. Our current inability to accurately assess long-term drinking behaviors is an important obstacle to its diagnosis and treatment. Biomarkers for chronic alcohol consumption have made a number of important advances but have yet to become highly accurate and as accepted as objective tests for other diseases. Thus, there is a crucial need for the development of more sensitive and specific markers of alcohol abuse. Recent advancements in proteomic technologies have greatly increased the potential for alcohol abuse biomarker discovery. Here, the authors review established and novel protein biomarkers for long-term alcohol consumption and the proteomic technologies that have been used in their study. PMID:22967079

Multiplexed Post-Experimental Monoisotopic Mass Refinement ( m PE-MMR) to Increase Sensitivity and Accuracy in Peptide Identifications from Tandem Mass Spectra of Cofragmentation

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

Madar, Inamul Hasan; Ko, Seung-Ik; Kim, Hokeun

Mass spectrometry (MS)-based proteomics, which uses high-resolution hybrid mass spectrometers such as the quadrupole-orbitrap mass spectrometer, can yield tens of thousands of tandem mass (MS/MS) spectra of high resolution during a routine bottom-up experiment. Despite being a fundamental and key step in MS-based proteomics, the accurate determination and assignment of precursor monoisotopic masses to the MS/MS spectra remains difficult. The difficulties stem from imperfect isotopic envelopes of precursor ions, inaccurate charge states for precursor ions, and cofragmentation. We describe a composite method of utilizing MS data to assign accurate monoisotopic masses to MS/MS spectra, including those subject to cofragmentation. Themore » method, “multiplexed post-experiment monoisotopic mass refinement” (mPE-MMR), consists of the following: multiplexing of precursor masses to assign multiple monoisotopic masses of cofragmented peptides to the corresponding multiplexed MS/MS spectra, multiplexing of charge states to assign correct charges to the precursor ions of MS/ MS spectra with no charge information, and mass correction for inaccurate monoisotopic peak picking. When combined with MS-GF+, a database search algorithm based on fragment mass difference, mPE-MMR effectively increases both sensitivity and accuracy in peptide identification from complex high-throughput proteomics data compared to conventional methods.« 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

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

The Pfam protein families database: towards a more sustainable future.

PubMed

Finn, Robert D; Coggill, Penelope; Eberhardt, Ruth Y; Eddy, Sean R; Mistry, Jaina; Mitchell, Alex L; Potter, Simon C; Punta, Marco; Qureshi, Matloob; Sangrador-Vegas, Amaia; Salazar, Gustavo A; Tate, John; Bateman, Alex

2016-01-04

In the last two years the Pfam database (http://pfam.xfam.org) has undergone a substantial reorganisation to reduce the effort involved in making a release, thereby permitting more frequent releases. Arguably the most significant of these changes is that Pfam is now primarily based on the UniProtKB reference proteomes, with the counts of matched sequences and species reported on the website restricted to this smaller set. Building families on reference proteomes sequences brings greater stability, which decreases the amount of manual curation required to maintain them. It also reduces the number of sequences displayed on the website, whilst still providing access to many important model organisms. Matches to the full UniProtKB database are, however, still available and Pfam annotations for individual UniProtKB sequences can still be retrieved. Some Pfam entries (1.6%) which have no matches to reference proteomes remain; we are working with UniProt to see if sequences from them can be incorporated into reference proteomes. Pfam-B, the automatically-generated supplement to Pfam, has been removed. The current release (Pfam 29.0) includes 16 295 entries and 559 clans. The facility to view the relationship between families within a clan has been improved by the introduction of a new tool. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Simultaneous Proteomic Discovery and Targeted Monitoring using Liquid Chromatography, Ion Mobility Spectrometry, and Mass Spectrometry

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

Burnum-Johnson, Kristin E.; Nie, Song; Casey, Cameron P.

Current proteomics approaches are comprised of both broad discovery measurements as well as more quantitative targeted measurements. These two different measurement types are used to initially identify potentially important proteins (e.g., candidate biomarkers) and then enable improved quantification for a limited number of selected proteins. However, both approaches suffer from limitations, particularly the lower sensitivity, accuracy, and quantitation precision for discovery approaches compared to targeted approaches, and the limited proteome coverage provided by targeted approaches. Herein, we describe a new proteomics approach that allows both discovery and targeted monitoring (DTM) in a single analysis using liquid chromatography, ion mobility spectrometrymore » and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled peptides for target ions are spiked into tryptic digests and both the labeled and unlabeled peptides are broadly detected using LC-IMS-MS instrumentation, allowing the benefits of discovery and targeted approaches. To understand the possible improvement of the DTM approach, it was compared to LC-MS broad measurements using an accurate mass and time tag database and selected reaction monitoring (SRM) targeted measurements. The DTM results yielded greater peptide/protein coverage and a significant improvement in the detection of lower abundance species compared to LC-MS discovery measurements. DTM was also observed to have similar detection limits as SRM for the targeted measurements indicating its potential for combining the discovery and targeted approaches.« less

Proteomic and genomic studies of non-alcoholic fatty liver disease - clues in the pathogenesis

PubMed Central

Lim, Jun Wei; Dillon, John; Miller, Michael

2014-01-01

Non-alcoholic fatty liver disease (NAFLD) is a widely prevalent hepatic disorder that covers wide spectrum of liver pathology. NAFLD is strongly associated with liver inflammation, metabolic hyperlipidaemia and insulin resistance. Frequently, NAFLD has been considered as the hepatic manifestation of metabolic syndrome. The pathophysiology of NAFLD has not been fully elucidated. Some patients can remain in the stage of simple steatosis, which generally is a benign condition; whereas others can develop liver inflammation and progress into non-alcoholic steatohepatitis, fibrosis, cirrhosis and hepatocellular carcinoma. The mechanism behind the progression is still not fully understood. Much ongoing proteomic researches have focused on discovering the unbiased circulating biochemical markers to allow early detection and treatment of NAFLD. Comprehensive genomic studies have also begun to provide new insights into the gene polymorphism to understand patient-disease variations. Therefore, NAFLD is considered a complex and mutifactorial disease phenotype resulting from environmental exposures acting on a susceptible polygenic background. This paper reviewed the current status of proteomic and genomic studies that have contributed to the understanding of NAFLD pathogenesis. For proteomics section, this review highlighted functional proteins that involved in: (1) transportation; (2) metabolic pathway; (3) acute phase reaction; (4) anti-inflammatory; (5) extracellular matrix; and (6) immune system. In the genomic studies, this review will discuss genes which involved in: (1) lipolysis; (2) adipokines; and (3) cytokines production. PMID:25024592

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.

Comparative Proteomics Reveals Novel Components at the Plasma Membrane of Differentiated HepaRG Cells and Different Distribution in Hepatocyte- and Biliary-Like Cells

PubMed Central

Woods, Alisa G.; Lazar, Catalin; Radu, Gabriel L.; Darie, Costel C.; Branza-Nichita, Norica

2013-01-01

Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitro, thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells. PMID:23977166

Image analysis tools and emerging algorithms for expression proteomics

PubMed Central

English, Jane A.; Lisacek, Frederique; Morris, Jeffrey S.; Yang, Guang-Zhong; Dunn, Michael J.

2012-01-01

Since their origins in academic endeavours in the 1970s, computational analysis tools have matured into a number of established commercial packages that underpin research in expression proteomics. In this paper we describe the image analysis pipeline for the established 2-D Gel Electrophoresis (2-DE) technique of protein separation, and by first covering signal analysis for Mass Spectrometry (MS), we also explain the current image analysis workflow for the emerging high-throughput ‘shotgun’ proteomics platform of Liquid Chromatography coupled to MS (LC/MS). The bioinformatics challenges for both methods are illustrated and compared, whilst existing commercial and academic packages and their workflows are described from both a user’s and a technical perspective. Attention is given to the importance of sound statistical treatment of the resultant quantifications in the search for differential expression. Despite wide availability of proteomics software, a number of challenges have yet to be overcome regarding algorithm accuracy, objectivity and automation, generally due to deterministic spot-centric approaches that discard information early in the pipeline, propagating errors. We review recent advances in signal and image analysis algorithms in 2-DE, MS, LC/MS and Imaging MS. Particular attention is given to wavelet techniques, automated image-based alignment and differential analysis in 2-DE, Bayesian peak mixture models and functional mixed modelling in MS, and group-wise consensus alignment methods for LC/MS. PMID:21046614

Developmental and Subcellular Organization of Single-Cell C₄ Photosynthesis in Bienertia sinuspersici Determined by Large-Scale Proteomics and cDNA Assembly from 454 DNA Sequencing.

PubMed

Offermann, Sascha; Friso, Giulia; Doroshenk, Kelly A; Sun, Qi; Sharpe, Richard M; Okita, Thomas W; Wimmer, Diana; Edwards, Gerald E; van Wijk, Klaas J

2015-05-01

Kranz C4 species strictly depend on separation of primary and secondary carbon fixation reactions in different cell types. In contrast, the single-cell C4 (SCC4) species Bienertia sinuspersici utilizes intracellular compartmentation including two physiologically and biochemically different chloroplast types; however, information on identity, localization, and induction of proteins required for this SCC4 system is currently very limited. In this study, we determined the distribution of photosynthesis-related proteins and the induction of the C4 system during development by label-free proteomics of subcellular fractions and leaves of different developmental stages. This was enabled by inferring a protein sequence database from 454 sequencing of Bienertia cDNAs. Large-scale proteome rearrangements were observed as C4 photosynthesis developed during leaf maturation. The proteomes of the two chloroplasts are different with differential accumulation of linear and cyclic electron transport components, primary and secondary carbon fixation reactions, and a triose-phosphate shuttle that is shared between the two chloroplast types. This differential protein distribution pattern suggests the presence of a mRNA or protein-sorting mechanism for nuclear-encoded, chloroplast-targeted proteins in SCC4 species. The combined information was used to provide a comprehensive model for NAD-ME type carbon fixation in SCC4 species.

SwissPalm: Protein Palmitoylation database.

PubMed

Blanc, Mathieu; David, Fabrice; Abrami, Laurence; Migliozzi, Daniel; Armand, Florence; Bürgi, Jérôme; van der Goot, Françoise Gisou

2015-01-01

Protein S-palmitoylation is a reversible post-translational modification that regulates many key biological processes, although the full extent and functions of protein S-palmitoylation remain largely unexplored. Recent developments of new chemical methods have allowed the establishment of palmitoyl-proteomes of a variety of cell lines and tissues from different species.  As the amount of information generated by these high-throughput studies is increasing, the field requires centralization and comparison of this information. Here we present SwissPalm ( http://swisspalm.epfl.ch), our open, comprehensive, manually curated resource to study protein S-palmitoylation. It currently encompasses more than 5000 S-palmitoylated protein hits from seven species, and contains more than 500 specific sites of S-palmitoylation. SwissPalm also provides curated information and filters that increase the confidence in true positive hits, and integrates predictions of S-palmitoylated cysteine scores, orthologs and isoform multiple alignments. Systems analysis of the palmitoyl-proteome screens indicate that 10% or more of the human proteome is susceptible to S-palmitoylation. Moreover, ontology and pathway analyses of the human palmitoyl-proteome reveal that key biological functions involve this reversible lipid modification. Comparative analysis finally shows a strong crosstalk between S-palmitoylation and other post-translational modifications. Through the compilation of data and continuous updates, SwissPalm will provide a powerful tool to unravel the global importance of protein S-palmitoylation.

SwissPalm: Protein Palmitoylation database

PubMed Central

Abrami, Laurence; Migliozzi, Daniel; Armand, Florence; Bürgi, Jérôme; van der Goot, Françoise Gisou

2015-01-01

Protein S-palmitoylation is a reversible post-translational modification that regulates many key biological processes, although the full extent and functions of protein S-palmitoylation remain largely unexplored. Recent developments of new chemical methods have allowed the establishment of palmitoyl-proteomes of a variety of cell lines and tissues from different species.  As the amount of information generated by these high-throughput studies is increasing, the field requires centralization and comparison of this information. Here we present SwissPalm ( http://swisspalm.epfl.ch), our open, comprehensive, manually curated resource to study protein S-palmitoylation. It currently encompasses more than 5000 S-palmitoylated protein hits from seven species, and contains more than 500 specific sites of S-palmitoylation. SwissPalm also provides curated information and filters that increase the confidence in true positive hits, and integrates predictions of S-palmitoylated cysteine scores, orthologs and isoform multiple alignments. Systems analysis of the palmitoyl-proteome screens indicate that 10% or more of the human proteome is susceptible to S-palmitoylation. Moreover, ontology and pathway analyses of the human palmitoyl-proteome reveal that key biological functions involve this reversible lipid modification. Comparative analysis finally shows a strong crosstalk between S-palmitoylation and other post-translational modifications. Through the compilation of data and continuous updates, SwissPalm will provide a powerful tool to unravel the global importance of protein S-palmitoylation. PMID:26339475

Genome-wide identification of the subcellular localization of the Escherichia coli B proteome using experimental and computational methods.

PubMed

Han, Mee-Jung; Yun, Hongseok; Lee, Jeong Wook; Lee, Yu Hyun; Lee, Sang Yup; Yoo, Jong-Shin; Kim, Jin Young; Kim, Jihyun F; Hur, Cheol-Goo

2011-04-01

Escherichia coli K-12 and B strains have most widely been employed for scientific studies as well as industrial applications. Recently, the complete genome sequences of two representative descendants of E. coli B strains, REL606 and BL21(DE3), have been determined. Here, we report the subproteome reference maps of E. coli B REL606 by analyzing cytoplasmic, periplasmic, inner and outer membrane, and extracellular proteomes based on the genome information using experimental and computational approaches. Among the total of 3487 spots, 651 proteins including 410 non-redundant proteins were identified and characterized by 2-DE and LC-MS/MS; they include 440 cytoplasmic, 45 periplasmic, 50 inner membrane, 61 outer membrane, and 55 extracellular proteins. In addition, subcellular localizations of all 4205 ORFs of E. coli B were predicted by combined computational prediction methods. The subcellular localizations of 1812 (43.09%) proteins of currently unknown function were newly assigned. The results of computational prediction were also compared with the experimental results, showing that overall precision and recall were 92.16 and 92.16%, respectively. This work represents the most comprehensive analyses of the subproteomes of E. coli B, and will be useful as a reference for proteome profiling studies under various conditions. The complete proteome data are available online (http://ecolib.kaist.ac.kr). Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Spatially resolved proteome mapping of laser capture microdissected tissue with automated sample transfer to nanodroplets.

PubMed

Zhu, Ying; Dou, Maowei; Piehowski, Paul D; Liang, Yiran; Wang, Fangjun; Chu, Rosalie K; Chrisler, Will; Smith, Jordan N; Schwarz, Kaitlynn C; Shen, Yufeng; Shukla, Anil K; Moore, Ronald J; Smith, Richard D; Qian, Wei-Jun; Kelly, Ryan T

2018-06-24

Current mass spectrometry (MS)-based proteomics approaches are ineffective for mapping protein expression in tissue sections with high spatial resolution due to the limited overall sensitivity of conventional workflows. Here we report an integrated and automated method to advance spatially resolved proteomics by seamlessly coupling laser capture microdissection (LCM) with a recently developed nanoliter-scale sample preparation system termed nanoPOTS (Nanodroplet Processing in One pot for Trace Samples). The workflow is enabled by prepopulating nanowells with DMSO, which serves as a sacrificial capture liquid for microdissected tissues. The DMSO droplets efficiently collect laser-pressure catapulted LCM tissues as small as 20 µm in diameter with success rates >87%. We also demonstrate that tissue treatment with DMSO can significantly improve proteome coverage, likely due to its ability to dissolve lipids from tissue and enhance protein extraction efficiency. The LCM-nanoPOTS platform was able to identify 180, 695, and 1827 protein groups on average from 12-µm-thick rat brain cortex tissue sections with diameters of 50, 100, and 200 µm, respectively. We also analyzed 100-µm-diameter sections corresponding to 10-18 cells from three different regions of rat brain and comparatively quantified ~1000 proteins, demonstrating the potential utility for high-resolution spatially resolved mapping of protein expression in tissues. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Sensitivity and specificity: twin goals of proteomics assays. Can they be combined?

PubMed

Wilson, Robert

2013-04-01

A major ambition of proteomics is the provision of assays that can diagnose disease and monitor therapies. These assays are required to be sensitive and specific for individual proteins, and in most cases to quantify more than one protein in the same sample. The two main technologies currently used for proteomics assays are based on mass spectrometry and panels of affinity molecules such as antibodies. In the first part of this review the most sensitive existing assays based on these technologies are described and compared with the gold standard of ELISA. Analytical sensitivity is defined and related to the limit of detection, and analytical specificity is defined and shown to depend on molecular proofreading steps, similar to those applied in living systems whenever there is a need for high fidelity. It is shown that at present neither mass spectrometry nor panels of affinity molecules offer the necessary combination of sensitivity and specificity required for multiplexed assays. In the second part of this review the growing numbers of assays that use additional proofreading steps to combine sensitivity with specificity are described. These include assays based on proximity ligation and slow off-rate modified aptamers. Finally the review considers what improvements might be possible in the near future, and concludes that further development of proteomics assays incorporating advanced proofreading steps are most likely to provide the necessary combination of sensitivity and specificity, without incurring high development costs.

Comparative proteomics reveals novel components at the plasma membrane of differentiated HepaRG cells and different distribution in hepatocyte- and biliary-like cells.

PubMed

Petrareanu, Catalina; Macovei, Alina; Sokolowska, Izabela; Woods, Alisa G; Lazar, Catalin; Radu, Gabriel L; Darie, Costel C; Branza-Nichita, Norica

2013-01-01

Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitro, thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells.

Proteome Analysis Using Isobaric Tags for Relative and Absolute Analysis Quantitation (iTRAQ) Reveals Alterations in Stress-Induced Dysfunctional Chicken Muscle.

PubMed

Xing, Tong; Wang, Chong; Zhao, Xue; Dai, Chen; Zhou, Guanghong; Xu, Xinglian

2017-04-05

The current study was designed to investigate changes in the protein profiles of pale, soft, and exudative (PSE)-like muscles of broilers subjected to transportation under high-temperature conditions, using isobaric tags for relative and absolute analysis quantitation (iTRAQ). Arbor Acres chickens (n = 112) were randomly divided into two treatments: unstressed control (CON) and 0.5 h of transport (T). Birds were transported according to a designed protocol. Pectoralis major (PM) muscle samples in the T group were collected and classified as normal (T-NOR) or PSE-like (T-PSE). Plasma activities of stress indicators, muscle microstructure, and proteome were measured. Results indicated that broilers in the T-PSE group exhibited higher activities of plasma stress indicators. The microstructure of T-PSE group showed a looser network and larger intercellular spaces in comparison to the other groups. Proteomic analysis, based on iTRAQ, revealed 29 differentially expressed proteins in the T-NOR and T-PSE groups that were involved in protein turnover, signal transduction, stress and defense, calcium handling, cell structure, and metabolism. In particular, proteins relating to the glycolysis pathway, calcium signaling, and molecular chaperones exhibited significant differences that may contribute to the inferior post-mortem meat quality. Overall, the proteomic results provide a further understanding of the mechanism of meat quality changes in response to stress.

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.

Salivary Cystatins: Exploring New Post-Translational Modifications and Polymorphisms by Top-Down High-Resolution Mass Spectrometry.

PubMed

Manconi, Barbara; Liori, Barbara; Cabras, Tiziana; Vincenzoni, Federica; Iavarone, Federica; Castagnola, Massimo; Messana, Irene; Olianas, Alessandra

2017-11-03

Cystatins are a complex family of cysteine peptidase inhibitors. In the present study, various proteoforms of cystatin A, cystatin B, cystatin S, cystatin SN, and cystatin SA were detected in the acid-soluble fraction of human saliva and characterized by a top-down HPLC-ESI-MS approach. Proteoforms of cystatin D were also detected and characterized by an integrated top-down and bottom-up strategy. The proteoforms derive from coding sequence polymorphisms and post-translational modifications, in particular, phosphorylation, N-terminal processing, and oxidation. This study increases the current knowledge of salivary cystatin proteoforms and provides the basis to evaluate possible qualitative/quantitative variations of these proteoforms in different pathological states and reveal new potential salivary biomarkers of disease. Data are available via ProteomeXchange with identifier PXD007170.

ECOTOXICOGENOMICS: LINKAGES BETWEEN EXPOSURE AND EFFECTS IN ASSESSING RISKS OF AQUATIC CONTAMINANTS TO FISH

EPA Science Inventory

Understanding the biological effects of exposures to chemicals in the environment relies on classical and emerging technologies in the areas of genomics, proteomics, and metabonomics. Linkages between the historical and newer toxicological tools are currently being developed in o...

The contribution of transcriptomic and proteomic analysis in elucidating stress adaptation responses of Listeria monocytogenes

USDA-ARS?s Scientific Manuscript database

The foodborne transmission of Listeria monocytogenes requires physiological adaptation to various conditions, including the cold, osmotic, heat, acid, alkaline, and oxidative stresses, associated with food hygiene, processing, and preservation measures. We review the current knowledge on the molecul...

Proteomic analysis of Mycoplasma gallisepticum vaccine strain F

USDA-ARS?s Scientific Manuscript database

The persistence and displacement abilities of the Mycoplasma gallisepticum vaccine strain F (F-strain) are well documented. Understanding the mechanism(s) of colonization and persistence of F-strain will aid in the current intervention strategies to diagnose and control MG infections in poultry. In ...

PROTEOMICS IN ECOTOXICOLOGY: PROTEIN EXPRESSION PROFILING TO SCREEN CHEMICALS FOR ENDOCRINE ACTIVITY

EPA Science Inventory

Abstract for poster.

Current endocrine testing methods are animal intensive and lack the throughput necessary to screen large numbers of environmental chemicals for adverse effects. In this study, Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry...

"Omics" of Selenium Biology: A Prospective Study of Plasma Proteome Network Before and After Selenized-Yeast Supplementation in Healthy Men.

PubMed

Sinha, Indu; Karagoz, Kubra; Fogle, Rachel L; Hollenbeak, Christopher S; Zea, Arnold H; Arga, Kazim Y; Stanley, Anne E; Hawkes, Wayne C; Sinha, Raghu

2016-04-01

Low selenium levels have been linked to a higher incidence of cancer and other diseases, including Keshan, Chagas, and Kashin-Beck, and insulin resistance. Additionally, muscle and cardiovascular disorders, immune dysfunction, cancer, neurological disorders, and endocrine function have been associated with mutations in genes encoding for selenoproteins. Selenium biology is complex, and a systems biology approach to study global metabolomics, genomics, and/or proteomics may provide important clues to examining selenium-responsive markers in circulation. In the current investigation, we applied a global proteomics approach on plasma samples collected from a previously conducted, double-blinded placebo controlled clinical study, where men were supplemented with selenized-yeast (Se-Yeast; 300 μg/day, 3.8 μmol/day) or placebo-yeast for 48 weeks. Proteomic analysis was performed by iTRAQ on 8 plasma samples from each arm at baseline and 48 weeks. A total of 161 plasma proteins were identified in both arms. Twenty-two proteins were significantly altered following Se-Yeast supplementation and thirteen proteins were significantly changed after placebo-yeast supplementation in healthy men. The differentially expressed proteins were involved in complement and coagulation pathways, immune functions, lipid metabolism, and insulin resistance. Reconstruction and analysis of protein-protein interaction network around selected proteins revealed several hub proteins. One of the interactions suggested by our analysis, PHLD-APOA4, which is involved in insulin resistance, was subsequently validated by Western blot analysis. Our systems approach illustrates a viable platform for investigating responsive proteomic profile in 'before and after' condition following Se-Yeast supplementation. The nature of proteins identified suggests that selenium may play an important role in complement and coagulation pathways, and insulin resistance.

Proteomics: a tool to develop novel diagnostic methods and unravel molecular mechanisms of pediatric diseases.

PubMed

Torres-Arroyo, Angélica; Ruiz-Lara, Arturo; Castillo-Villanueva, Adriana; Méndez-Cruz, Sara Teresa; Espinosa-Padilla, Sara Elvia; Espinosa-Rosales, Francisco Javier; Zarate-Mondragón, Flora; Cervantes-Bustamante, Roberto; Bosch-Canto, Vanessa; Vizzuett-López, Iris; Ordaz-Fávila, Juan Carlos; Oria-Hernández, Jesús; Reyes-Vivas, Horacio

Proteomics is the study of the expression of changes and post-translational modifications (PTM) of proteins along a metabolic condition either normal or pathological. In the field of health, proteomics allows obtaining valuable data for treatment, diagnosis or pathophysiological mechanisms of different illnesses. To illustrate the aforementioned, we describe two projects currently being performed at the Instituto Nacional de Pediatría: The immuno-proteomic study of cow milk allergy and the Proteomic study of childhood cataract. Cow's milk proteins (CMP) are the first antigens to which infants are exposed and generate allergy in some of them. In Mexico, the incidence of CMP allergy has been estimated at 5-7%. Clinical manifestations include both gastrointestinal and extra-gastrointestinal symptoms, making its diagnosis extremely difficult. An inappropriate diagnosis affects the development and growth of children. The goals of the study are to identify the main immune-reactive CMP in Mexican pediatric population and to design more accurate diagnostic tools for this disease. Childhood cataract is a major ocular disease representing one of the main causes of blindness in infants; in developing countries, this disease promotes up to 27% of cases related to visual loss. From this group, it has been estimated that close to 60% of children do not survive beyond two years after vision lost. PTM have been pointed out as the main cause of protein precipitation at the crystalline and, consequently, clouding of this tissue. The study of childhood cataract represents an outstanding opportunity to identify the PTM associated to the cataract-genesis process. Copyright © 2017 Hospital Infantil de México Federico Gómez. Publicado por Masson Doyma México S.A. All rights reserved.

Comparative membrane proteomics analyses of breast cancer cell lines to understand the molecular mechanism of breast cancer brain metastasis.

PubMed

Peng, Wenjing; Zhang, Yu; Zhu, Rui; Mechref, Yehia

2017-09-01

Breast cancer is the leading type of cancer in women. Breast cancer brain metastasis is currently considered an issue of concern among breast cancer patients. Membrane proteins play important roles in breast cancer brain metastasis, involving cell adhesion and penetration of blood-brain barrier. To understand the mechanism of breast cancer brain metastasis, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed in conjunction with enrichment of membrane proteins to analyze the proteomes from five different breast cancer and a brain cancer cell lines. Quantitative proteomic data of all cell lines were compared with MDA-MB-231BR which is a brain seeking breast cancer cell line, thus representing brain metastasis characteristics. Label-free proteomics of the six cell lines facilitates the identification of 1238 proteins and the quantification of 899 proteins of which more than 70% were membrane proteins. Unsupervised principal component analysis (PCA) of the label-free proteomics data resulted in a distinct clustering of cell lines, suggesting quantitative differences in the expression of several proteins among the different cell lines. Unique protein expressions in 231BR were observed for 28 proteins. The up-regulation of STAU1, AT1B3, NPM1, hnRNP Q, and hnRNP K and the down-regulation of TUBB4B and TUBB5 were noted in 231BR relative to 231 (precursor cell lines from which 231BR is derived). These proteins might contribute to the breast cancer brain metastasis. Ingenuity pathway analysis (IPA) supported the great brain metastatic propensity of 231BR and suggested the importance of the up-regulation of integrin proteins and down-regulation of EPHA2 in brain metastasis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Surface analysis of Dicrocoelium dendriticum. The molecular characterization of exosomes reveals the presence of miRNAs.

PubMed

Bernal, Dolores; Trelis, Maria; Montaner, Sergio; Cantalapiedra, Fernando; Galiano, Alicia; Hackenberg, Michael; Marcilla, Antonio

2014-06-13

With the aim of characterizing the molecules involved in the interaction of Dicrocoelium dendriticum adults and the host, we have performed proteomic analyses of the external surface of the parasite using the currently available datasets including the transcriptome of the related species Echinostoma caproni. We have identified 182 parasite proteins on the outermost surface of D. dendriticum. The presence of exosome-like vesicles in the ESP of D. dendriticum and their components has also been characterized. Using proteomic approaches, we have characterized 84 proteins in these vesicles. Interestingly, we have detected miRNA in D. dendriticum exosomes, thus representing the first report of miRNA in helminth exosomes. In order to identify potential targets for intervention against parasitic helminths, we have analyzed the surface of the parasitic helminth Dicrocoelium dendriticum. Along with the proteomic analyses of the outermost layer of the parasite, our work describes the molecular characterization of the exosomes of D. dendriticum. Our proteomic data confirm the improvement of protein identification from "non-model organisms" like helminths, when using different search engines against a combination of available databases. In addition, this work represents the first report of miRNAs in parasitic helminth exosomes. These vesicles can pack specific proteins and RNAs providing stability and resistance to RNAse digestion in body fluids, and provide a way to regulate host-parasite interplay. The present data should provide a solid foundation for the development of novel methods to control this non-model organism and related parasites. This article is part of a Special Issue entitled: Proteomics of non-model organisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Nano-LC FTICR tandem mass spectrometry for top-down proteomics: routine baseline unit mass resolution of whole cell lysate proteins up to 72 kDa.

PubMed

Tipton, Jeremiah D; Tran, John C; Catherman, Adam D; Ahlf, Dorothy R; Durbin, Kenneth R; Lee, Ji Eun; Kellie, John F; Kelleher, Neil L; Hendrickson, Christopher L; Marshall, Alan G

2012-03-06

Current high-throughput top-down proteomic platforms provide routine identification of proteins less than 25 kDa with 4-D separations. This short communication reports the application of technological developments over the past few years that improve protein identification and characterization for masses greater than 25 kDa. Advances in separation science have allowed increased numbers of proteins to be identified, especially by nanoliquid chromatography (nLC) prior to mass spectrometry (MS) analysis. Further, a goal of high-throughput top-down proteomics is to extend the mass range for routine nLC MS analysis up to 80 kDa because gene sequence analysis predicts that ~70% of the human proteome is transcribed to be less than 80 kDa. Normally, large proteins greater than 50 kDa are identified and characterized by top-down proteomics through fraction collection and direct infusion at relatively low throughput. Further, other MS-based techniques provide top-down protein characterization, however at low resolution for intact mass measurement. Here, we present analysis of standard (up to 78 kDa) and whole cell lysate proteins by Fourier transform ion cyclotron resonance mass spectrometry (nLC electrospray ionization (ESI) FTICR MS). The separation platform reduced the complexity of the protein matrix so that, at 14.5 T, proteins from whole cell lysate up to 72 kDa are baseline mass resolved on a nano-LC chromatographic time scale. Further, the results document routine identification of proteins at improved throughput based on accurate mass measurement (less than 10 ppm mass error) of precursor and fragment ions for proteins up to 50 kDa.

Characterization and quantification of proteins secreted by single human embryos prior to implantation.

PubMed

Poli, Maurizio; Ori, Alessandro; Child, Tim; Jaroudi, Souraya; Spath, Katharina; Beck, Martin; Wells, Dagan

2015-11-01

The use of in vitro fertilization (IVF) has revolutionized the treatment of infertility and is now responsible for 1-5% of all births in industrialized countries. During IVF, it is typical for patients to generate multiple embryos. However, only a small proportion of them possess the genetic and metabolic requirements needed in order to produce a healthy pregnancy. The identification of the embryo with the greatest developmental capacity represents a major challenge for fertility clinics. Current methods for the assessment of embryo competence are proven inefficient, and the inadvertent transfer of non-viable embryos is the principal reason why most IVF treatments (approximately two-thirds) end in failure. In this study, we investigate how the application of proteomic measurements could improve success rates in clinical embryology. We describe a procedure that allows the identification and quantification of proteins of embryonic origin, present in attomole concentrations in the blastocoel, the enclosed fluid-filled cavity that forms within 5-day-old human embryos. By using targeted proteomics, we demonstrate the feasibility of quantifying multiple proteins in samples derived from single blastocoels and that such measurements correlate with aspects of embryo viability, such as chromosomal (ploidy) status. This study illustrates the potential of high-sensitivity proteomics to measure clinically relevant biomarkers in minute samples and, more specifically, suggests that key aspects of embryo competence could be measured using a proteomic-based strategy, with negligible risk of harm to the living embryo. Our work paves the way for the development of "next-generation" embryo competence assessment strategies, based on functional proteomics. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

Proteomic profiling of the planarian Schmidtea mediterranea and its mucous reveals similarities with human secretions and those predicted for parasitic flatworms.

PubMed

Bocchinfuso, Donald G; Taylor, Paul; Ross, Eric; Ignatchenko, Alex; Ignatchenko, Vladimir; Kislinger, Thomas; Pearson, Bret J; Moran, Michael F

2012-09-01

The freshwater planarian Schmidtea mediterranea has been used in research for over 100 years, and is an emerging stem cell model because of its capability of regenerating large portions of missing body parts. Exteriorly, planarians are covered in mucous secretions of unknown composition, implicated in locomotion, predation, innate immunity, and substrate adhesion. Although the planarian genome has been sequenced, it remains mostly unannotated, challenging both genomic and proteomic analyses. The goal of the current study was to annotate the proteome of the whole planarian and its mucous fraction. The S. mediterranea proteome was analyzed via mass spectrometry by using multidimensional protein identification technology with whole-worm tryptic digests. By using a proteogenomics approach, MS data were searched against an in silico translated planarian transcript database, and by using the Swiss-Prot BLAST algorithm to identify proteins similar to planarian queries. A total of 1604 proteins were identified. The mucous subproteome was defined through analysis of a mucous trail fraction and an extract obtained by treating whole worms with the mucolytic agent N-acetylcysteine. Gene Ontology analysis confirmed that the mucous fractions were enriched with secreted proteins. The S. mediterranea proteome is highly similar to that predicted for the trematode Schistosoma mansoni associated with intestinal schistosomiasis, with the mucous subproteome particularly highly conserved. Remarkably, orthologs of 119 planarian mucous proteins are present in human mucosal secretions and tear fluid. We suggest planarians have potential to be a model system for the characterization of mucous protein function and relevant to parasitic flatworm infections and diseases underlined by mucous aberrancies, such as cystic fibrosis, asthma, and other lung diseases.

The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics*

PubMed Central

Beck, Scarlet; Michalski, Annette; Raether, Oliver; Lubeck, Markus; Kaspar, Stephanie; Goedecke, Niels; Baessmann, Carsten; Hornburg, Daniel; Meier, Florian; Paron, Igor; Kulak, Nils A.; Cox, Juergen; Mann, Matthias

2015-01-01

Hybrid quadrupole time-of-flight (QTOF) mass spectrometry is one of the two major principles used in proteomics. Although based on simple fundamentals, it has over the last decades greatly evolved in terms of achievable resolution, mass accuracy, and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated liquid chromatography system achieved very narrow peptide elution peaks. The impact II is equipped with a new collision cell with both axial and radial ion ejection, more than doubling ion extraction at high tandem MS frequencies. The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222. We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell. Simulation and measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for QTOF data, improving absolute average mass deviations to better than 1.45 ppm. More than 4800 proteins can be identified in a single run of HeLa digest in a 90 min gradient. The workflow achieved high technical reproducibility (R2 > 0.99) and accurate fold change determination in spike-in experiments in complex mixtures. Using label-free quantification we rapidly quantified haploid against diploid yeast and characterized overall proteome differences in mouse cell lines originating from different tissues. Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum—the highest proteome coverage reported with a QTOF instrument so far. PMID:25991688

Proteomic analysis of Bombyx mori molting fluid: Insights into the molting process.

PubMed

Liu, Hua-Wei; Wang, Luo-Ling; Tang, Xin; Dong, Zhao-Ming; Guo, Peng-Chao; Zhao, Dong-Chao; Xia, Qing-You; Zhao, Ping

2018-02-20

Molting is an essential biological process occurring multiple times throughout the life cycle of most Ecdysozoa. Molting fluids accumulate and function in the exuvial space during the molting process. In this study, we used liquid chromatography-tandem mass spectrometry to investigate the molting fluids to analyze the molecular mechanisms of molting in the silkworm, Bombyx mori. In total, 375 proteins were identified in molting fluids from the silkworm at 14-16h before pupation and eclosion, including 12 chitin metabolism-related enzymes, 35 serine proteases, 15 peptidases, and 38 protease inhibitors. Gene ontology analysis indicated that "catalytic" constitutes the most enriched function in the molting fluid. Gene expression patterns and bioinformatic analyses suggested that numerous enzymes are involved in the degradation of cuticle proteins and chitin. Protein-protein interaction network and activity analyses showed that protease inhibitors are involved in the regulation of multiple pathways in molting fluid. Additionally, many immune-related proteins may be involved in the immune defense during molting. These results provide a comprehensive proteomic insight into proteolytic enzymes and protease inhibitors in molting fluid, and will likely improve the current understanding of physiological processes in insect molting. Insect molting constitutes a dynamic physiological process. To better understand this process, we used LC-MS/MS to investigate the proteome of silkworm molting fluids and identified key proteins involved in silkworm molting. The biological processes of the old cuticle degradation pathway and immune defense response were analyzed in the proteome of silkworm molting fluid. We report that protease inhibitors serve as key factors in the regulation of the molting process. The proteomic results provide new insight into biological molting processes in insects. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparative Proteomic Analysis of Whole-Gut Lavage Fluid and Pancreatic Juice Reveals a Less Invasive Method of Sampling Pancreatic Secretions

PubMed Central

Rocker, Jana M; Tan, Marcus C; Thompson, Lee W; Contreras, Carlo M; DiPalma, Jack A; Pannell, Lewis K

2016-01-01

OBJECTIVES: There are currently no reliable, non-invasive screening tests for pancreatic ductal adenocarcinoma. The fluid secreted from the pancreatic ductal system (“pancreatic juice”) has been well-studied as a potential source of cancer biomarkers. However, it is invasive to collect. We recently observed that the proteomic profile of intestinal effluent from the bowel in response to administration of an oral bowel preparation solution (also known as whole-gut lavage fluid, WGLF) contains large amounts of pancreas-derived proteins. We therefore hypothesized that the proteomic profile is similar to that of pancreatic juice. In this study, we compared the proteomic profiles of 77 patients undergoing routine colonoscopy with the profiles of 19 samples of pure pancreatic juice collected during surgery. METHODS: WGLF was collected from patients undergoing routine colonoscopy, and pancreatic juice was collected from patients undergoing pancreatic surgery. Protein was isolated from both samples using an optimized method and analyzed by LC-MS/MS. Identified proteins were compared between samples and groups to determine similarity of the two fluids. We then compared our results with literature reports of pancreatic juice-based studies to determine similarity. RESULTS: We found 104 proteins in our pancreatic juice samples, of which 90% were also found in our WGLF samples. The majority (67%) of the total proteins found in the WGLF were common to pancreatic juice, with intestine-specific proteins making up a smaller proportion. CONCLUSIONS: WGLF and pancreatic juice appear to have similar proteomic profiles. This supports the notion that WGLF is a non-invasive, surrogate bio-fluid for pancreatic juice. Further studies are required to further elucidate its role in the diagnosis of pancreatic cancer. PMID:27228405

Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics

PubMed Central

Bennett, Eric J.; Rush, John; Gygi, Steven P.; Harper, J. Wade

2010-01-01

Dynamic reorganization of signaling systems frequently accompany pathway perturbations, yet quantitative studies of network remodeling by pathway stimuli are lacking. Here, we report the development of a quantitative proteomics platform centered on multiplex Absolute Quantification (AQUA) technology to elucidate the architecture of the cullin-RING ubiquitin ligase (CRL) network and to evaluate current models of dynamic CRL remodeling. Current models suggest that CRL complexes are controlled by cycles of CRL deneddylation and CAND1 binding. Contrary to expectations, acute CRL inhibition with MLN4924, an inhibitor of the NEDD8-activating enzyme, does not result in a global reorganization of the CRL network. Examination of CRL complex stoichiometry reveals that, independent of cullin neddylation, a large fraction of cullins are assembled with adaptor modules while only a small fraction are associated with CAND1. These studies suggest an alternative model of CRL dynamicity where the abundance of adaptor modules, rather than cycles of neddylation and CAND1 binding, drives CRL network organization. PMID:21145461

Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics.

PubMed

Bennett, Eric J; Rush, John; Gygi, Steven P; Harper, J Wade

2010-12-10

Dynamic reorganization of signaling systems frequently accompanies pathway perturbations, yet quantitative studies of network remodeling by pathway stimuli are lacking. Here, we report the development of a quantitative proteomics platform centered on multiplex absolute quantification (AQUA) technology to elucidate the architecture of the cullin-RING ubiquitin ligase (CRL) network and to evaluate current models of dynamic CRL remodeling. Current models suggest that CRL complexes are controlled by cycles of CRL deneddylation and CAND1 binding. Contrary to expectations, acute CRL inhibition with MLN4924, an inhibitor of the NEDD8-activating enzyme, does not result in a global reorganization of the CRL network. Examination of CRL complex stoichiometry reveals that, independent of cullin neddylation, a large fraction of cullins are assembled with adaptor modules, whereas only a small fraction are associated with CAND1. These studies suggest an alternative model of CRL dynamicity where the abundance of adaptor modules, rather than cycles of neddylation and CAND1 binding, drives CRL network organization. Copyright © 2010 Elsevier Inc. All rights reserved.

Gene and Protein Expression following Exposure to Radiofrequency Fields from Mobile Phones

PubMed Central

Vanderstraeten, Jacques; Verschaeve, Luc

2008-01-01

Background Since 1999, several articles have been published on genome-wide and/or proteome-wide response after exposure to radiofrequency (RF) fields whose signal and intensities were similar to or typical of those of currently used mobile telephones. These studies were performed using powerful high-throughput screening techniques (HTSTs) of transcriptomics and/or proteomics, which allow for the simultaneous screening of the expression of thousands of genes or proteins. Objectives We reviewed these HTST-based studies and compared the results with currently accepted concepts about the effects of RF fields on gene expression. In this article we also discuss these last in light of the recent concept of microwave-assisted chemistry. Discussion To date, the results of HTST-based studies of transcriptomics and/or proteomics after exposure to RF fields relevant to human exposure are still inconclusive, as most of the positive reports are flawed by methodologic imperfections or shortcomings. In addition, when positive findings were reported, no precise response pattern could be identified in a reproducible way. In particular, results from HTST studies tend to exclude the role of a cell stressor for exposure to RF fields at nonthermal intensities. However, on the basis of lessons from microwave-assisted chemistry, we can assume that RF fields might affect heat-sensitive gene or protein expression to an extent larger than would be predicted from temperature change only. But in all likelihood, this would concern intensities higher than those relevant to usual human exposure. Conclusions The precise role of transcriptomics and proteomics in the screening of bioeffects from exposure to RF fields from mobile phones is still uncertain in view of the lack of positively identified phenotypic change and the lack of theoretical, as well as experimental, arguments for specific gene and/or protein response patterns after this kind of exposure. PMID:18795152

Characterization of the Proteome of Cytoplasmic Lipid Droplets in Mouse Enterocytes after a Dietary Fat Challenge

PubMed Central

D’Aquila, Theresa; Sirohi, Devika; Grabowski, Jeffrey M.; Hedrick, Victoria E.; Paul, Lake N.; Greenberg, Andrew S.; Kuhn, Richard J.; Buhman, Kimberly K.

2015-01-01

Dietary fat absorption by the small intestine is a multistep process that regulates the uptake and delivery of essential nutrients and energy. One step of this process is the temporary storage of dietary fat in cytoplasmic lipid droplets (CLDs). The storage and mobilization of dietary fat is thought to be regulated by proteins that associate with the CLD; however, mechanistic details of this process are currently unknown. In this study we analyzed the proteome of CLDs isolated from enterocytes harvested from the small intestine of mice following a dietary fat challenge. In this analysis we identified 181 proteins associated with the CLD fraction, of which 37 are associated with known lipid related metabolic pathways. We confirmed the localization of several of these proteins on or around the CLD through confocal and electron microscopy, including perilipin 3, apolipoprotein A-IV, and acyl-CoA synthetase long-chain family member 5. The identification of the enterocyte CLD proteome provides new insight into potential regulators of CLD metabolism and the process of dietary fat absorption. PMID:25992653

A Researcher's Guide to Mass Spectrometry-Based Proteomics

PubMed Central

Savaryn, John P.; Toby, Timothy K.; Kelleher, Neil L.

2016-01-01

Mass spectrometry (MS) is widely recognized as a powerful analytical tool for molecular research. MS is used by researchers around the globe to identify, quantify, and characterize biomolecules like proteins from any number of biological conditions or sample types. As instrumentation has advanced, and with the coupling of liquid chromatography (LC) for high-throughput LC-MS/MS, a proteomics experiment measuring hundreds to thousands of proteins/protein groups is now commonplace. While expert practitioners who best understand the operation of LC-MS systems tend to have strong backgrounds in physics and engineering, consumers of proteomics data and technology are not exposed to the physio-chemical principles underlying the information they seek. Since articles and reviews tend not to focus on bridging this divide, our goal here is to span this gap and translate MS ion physics into language intuitive to the general reader active in basic or applied biomedical research. Here, we visually describe what happens to ions as they enter and move around inside a mass spectrometer. We describe basic MS principles, including electric current, ion optics, ion traps, quadrupole mass filters, and Orbitrap FT-analyzers. PMID:27553853