RESUMO
The inherent diversity of approaches in proteomics research has led to a wide range of software solutions for data analysis. These software solutions encompass multiple tools, each employing different algorithms for various tasks such as peptide-spectrum matching, protein inference, quantification, statistical analysis, and visualization. To enable an unbiased comparison of commonly used bottom-up label-free proteomics workflows, we introduce WOMBAT-P, a versatile platform designed for automated benchmarking and comparison. WOMBAT-P simplifies the processing of public data by utilizing the sample and data relationship format for proteomics (SDRF-Proteomics) as input. This feature streamlines the analysis of annotated local or public ProteomeXchange data sets, promoting efficient comparisons among diverse outputs. Through an evaluation using experimental ground truth data and a realistic biological data set, we uncover significant disparities and a limited overlap in the quantified proteins. WOMBAT-P not only enables rapid execution and seamless comparison of workflows but also provides valuable insights into the capabilities of different software solutions. These benchmarking metrics are a valuable resource for researchers in selecting the most suitable workflow for their specific data sets. The modular architecture of WOMBAT-P promotes extensibility and customization. The software is available at https://github.com/wombat-p/WOMBAT-Pipelines.
Assuntos
Benchmarking , Proteômica , Fluxo de Trabalho , Software , Proteínas , Análise de DadosRESUMO
INTRODUCTION: Continuous advances in mass spectrometry (MS) technologies have enabled deeper and more reproducible proteome characterization and a better understanding of biological systems when integrated with other 'omics data. Bioinformatic resources meeting the analysis requirements of increasingly complex MS-based proteomic data and associated multi-omic data are critically needed. These requirements included availability of software that would span diverse types of analyses, scalability for large-scale, compute-intensive applications, and mechanisms to ease adoption of the software. AREAS COVERED: The Galaxy ecosystem meets these requirements by offering a multitude of open-source tools for MS-based proteomics analyses and applications, all in an adaptable, scalable, and accessible computing environment. A thriving global community maintains these software and associated training resources to empower researcher-driven analyses. EXPERT OPINION: The community-supported Galaxy ecosystem remains a crucial contributor to basic biological and clinical studies using MS-based proteomics. In addition to the current status of Galaxy-based resources, we describe ongoing developments for meeting emerging challenges in MS-based proteomic informatics. We hope this review will catalyze increased use of Galaxy by researchers employing MS-based proteomics and inspire software developers to join the community and implement new tools, workflows, and associated training content that will add further value to this already rich ecosystem.
Assuntos
Proteômica , Humanos , Biologia Computacional/métodos , Espectrometria de Massas/métodos , Proteômica/métodos , SoftwareRESUMO
Proteo3Dnet is a web server dedicated to the analysis of mass spectrometry interactomics experiments. Given a flat list of proteins, its aim is to organize it in terms of structural interactions to provide a clearer overview of the data. This is achieved using three means: (i) the search for interologs with resolved structure available in the protein data bank, including cross-species remote homology search, (ii) the search for possibly weaker interactions mediated through Short Linear Motifs as predicted by ELM-a unique feature of Proteo3Dnet, (iii) the search for protein-protein interactions physically validated in the BioGRID database. The server then compiles this information and returns a graph of the identified interactions and details about the different searches. The graph can be interactively explored to understand the way the core complexes identified could interact. It can also suggest undetected partners to the experimentalists, or specific cases of conditionally exclusive binding. The interest of Proteo3Dnet, previously demonstrated for the difficult cases of the proteasome and pragmin complexes data is, here, illustrated in the context of yeast precursors to the small ribosomal subunits and the smaller interactome of 14-3-3zeta frequent interactors. The Proteo3Dnet web server is accessible at http://bioserv.rpbs.univ-paris-diderot.fr/services/Proteo3Dnet/.
Assuntos
Conformação Proteica , Mapeamento de Interação de Proteínas/métodos , Software , Proteínas 14-3-3/metabolismo , Internet , Espectrometria de Massas , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Proteômica , Subunidades Ribossômicas Menores de Eucariotos/metabolismoRESUMO
The study of proteins circulating in blood offers tremendous opportunities to diagnose, stratify, or possibly prevent diseases. With recent technological advances and the urgent need to understand the effects of COVID-19, the proteomic analysis of blood-derived serum and plasma has become even more important for studying human biology and pathophysiology. Here we provide views and perspectives about technological developments and possible clinical applications that use mass-spectrometry(MS)- or affinity-based methods. We discuss examples where plasma proteomics contributed valuable insights into SARS-CoV-2 infections, aging, and hemostasis and the opportunities offered by combining proteomics with genetic data. As a contribution to the Human Proteome Organization (HUPO) Human Plasma Proteome Project (HPPP), we present the Human Plasma PeptideAtlas build 2021-07 that comprises 4395 canonical and 1482 additional nonredundant human proteins detected in 240 MS-based experiments. In addition, we report the new Human Extracellular Vesicle PeptideAtlas 2021-06, which comprises five studies and 2757 canonical proteins detected in extracellular vesicles circulating in blood, of which 74% (2047) are in common with the plasma PeptideAtlas. Our overview summarizes the recent advances, impactful applications, and ongoing challenges for translating plasma proteomics into utility for precision medicine.
Assuntos
Proteoma , Proteômica/tendências , Envelhecimento/genética , COVID-19/genética , Bases de Dados de Proteínas , Hemostasia/genética , Humanos , Espectrometria de Massas , Proteoma/genéticaRESUMO
Acute liver injury (ALI) is a severe disorder resulting from excessive hepatocyte cell death, and frequently caused by acetaminophen intoxication. Clinical management of ALI progression is hampered by the dearth of blood biomarkers available. In this study, a bioinformatics workflow was developed to screen omics databases and identify potential biomarkers for hepatocyte cell death. Then, discovery proteomics was harnessed to select from among these candidates those that were specifically detected in the blood of acetaminophen-induced ALI patients. Among these candidates, the isoenzyme alcohol dehydrogenase 1B (ADH1B) was massively leaked into the blood. To evaluate ADH1B, we developed a targeted proteomics assay and quantified ADH1B in serum samples collected at different times from 17 patients admitted for acetaminophen-induced ALI. Serum ADH1B concentrations increased markedly during the acute phase of the disease, and dropped to undetectable levels during recovery. In contrast to alanine aminotransferase activity, the rapid drop in circulating ADH1B concentrations was followed by an improvement in the international normalized ratio (INR) within 10-48 h, and was associated with favorable outcomes. In conclusion, the combination of omics data exploration and proteomics revealed ADH1B as a new blood biomarker candidate that could be useful for the monitoring of acetaminophen-induced ALI.
Assuntos
Álcool Desidrogenase/sangue , Biomarcadores/sangue , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Proteômica/métodos , Acetaminofen/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/patologia , Cromatografia Líquida de Alta Pressão , Biologia Computacional , Humanos , Coeficiente Internacional Normatizado , Limite de Detecção , Espectrometria de Massas em TandemRESUMO
In the context of the Human Proteome Project, we built an inventory of 412 functionally unannotated human proteins for which experimental evidence at the protein level exists (uPE1) and which are highly expressed in tissues involved in human male reproduction. We implemented a strategy combining literature mining, bioinformatics tools to collate annotation and experimental information from specific molecular public resources, and efficient visualization tools to put these unknown proteins into their biological context (protein complexes, tissue and subcellular location, expression pattern). The gathered knowledge allowed pinpointing five uPE1 for which a function has recently been proposed and which should be updated in protein knowledge bases. Furthermore, this bioinformatics strategy allowed to build new functional hypotheses for five other uPE1s in link with phenotypic traits that are specific to male reproductive function such as ciliogenesis/flagellum formation in germ cells (CCDC112 and TEX9), chromatin remodeling (C3orf62) and spermatozoon maturation (CCDC183). We also discussed the enigmatic case of MAGEB proteins, a poorly documented cancer/testis antigen subtype. Tools used and computational outputs produced during this study are freely accessible via ProteoRE (http://www.proteore.org), a Galaxy-based instance, for reuse purposes. We propose these five uPE1s should be investigated in priority by expert laboratories and hope that this inventory and shared resources will stimulate the interest of the community of reproductive biology.
Assuntos
Proteoma , Proteômica , Biologia Computacional , Humanos , Bases de Conhecimento , Masculino , Proteoma/genética , ReproduçãoRESUMO
Protein-protein interactions play a major role in the molecular machinery of life, and various techniques such as AP-MS are dedicated to their identification. However, those techniques return lists of proteins devoid of organizational structure, not detailing which proteins interact with which others. Proposing a hierarchical view of the interactions between the members of the flat list becomes highly tedious for large data sets when done by hand. To help hierarchize this data, we introduce a new bioinformatics protocol that integrates information of the multimeric protein 3D structures available in the Protein Data Bank using remote homology detection, as well as information related to Short Linear Motifs and interaction data from the BioGRID. We illustrate on two unrelated use-cases of different complexity how our approach can be useful to decipher the network of interactions hidden in the list of input proteins, and how it provides added value compared to state-of-the-art resources such as Interactome3D or STRING. Particularly, we show the added value of using homology detection to distinguish between orthologs and paralogs, and to distinguish between core obligate and more facultative interactions. We also demonstrate the potential of considering interactions occurring through Short Linear Motifs.
Assuntos
Mapas de Interação de Proteínas , Proteômica , Biologia Computacional , Bases de Dados de Proteínas , Mapeamento de Interação de Proteínas , Proteínas/genética , Proteínas/metabolismoRESUMO
Secretome proteomics for the discovery of cancer biomarkers holds great potential to improve early cancer diagnosis. A knowledge-based approach relying on mechanistic criteria related to the type of cancer should help to identify candidates from available "omics" information. With the aim of accelerating the discovery process for novel biomarkers, a set of tools is developed and made available via a Galaxy-based instance to assist end-users biologists. These implemented tools proceed by a step-by-step strategy to mine transcriptomics and proteomics databases for information relating to tissue specificity, allow the selection of proteins that are part of the secretome, and combine this information with proteomics datasets to rank the most promising candidate biomarkers for early cancer diagnosis. Using pancreatic cancer as a case study, this strategy produces a list of 24 candidate biomarkers suitable for experimental assessment by MS-based proteomics. Among these proteins, three (SYCN, REG1B, and PRSS2) were previously reported as circulating candidate biomarkers of pancreatic cancer. Here, further refinement of this list allows to prioritize 14 candidate biomarkers along with their associated proteotypic peptides for further investigation, using targeted MS-based proteomics. The bioinformatics tools and the workflow implementing this strategy for the selection of candidate biomarkers are freely accessible at http://www.proteore.org.
Assuntos
Biomarcadores Tumorais/sangue , Detecção Precoce de Câncer , Neoplasias Pancreáticas/sangue , Proteômica/métodos , Biologia Computacional/métodos , Humanos , Neoplasias Pancreáticas/patologia , Proteoma/genética , Software , Fluxo de TrabalhoRESUMO
The Human Proteome Organization's (HUPO) Human Proteome Project (HPP) developed Mass Spectrometry (MS) Data Interpretation Guidelines that have been applied since 2016. These guidelines have helped ensure that the emerging draft of the complete human proteome is highly accurate and with low numbers of false-positive protein identifications. Here, we describe an update to these guidelines based on consensus-reaching discussions with the wider HPP community over the past year. The revised 3.0 guidelines address several major and minor identified gaps. We have added guidelines for emerging data independent acquisition (DIA) MS workflows and for use of the new Universal Spectrum Identifier (USI) system being developed by the HUPO Proteomics Standards Initiative (PSI). In addition, we discuss updates to the standard HPP pipeline for collecting MS evidence for all proteins in the HPP, including refinements to minimum evidence. We present a new plan for incorporating MassIVE-KB into the HPP pipeline for the next (HPP 2020) cycle in order to obtain more comprehensive coverage of public MS data sets. The main checklist has been reorganized under headings and subitems, and related guidelines have been grouped. In sum, Version 2.1 of the HPP MS Data Interpretation Guidelines has served well, and this timely update to version 3.0 will aid the HPP as it approaches its goal of collecting and curating MS evidence of translation and expression for all predicted â¼20â¯000 human proteins encoded by the human genome.
Assuntos
Guias como Assunto , Espectrometria de Massas/métodos , Proteoma , Processamento de Sinais Assistido por Computador , Humanos , Proteômica , Sociedades CientíficasRESUMO
BACKGROUND: Accurate structural annotation of genomes is still a challenge, despite the progress made over the past decade. The prediction of gene structure remains difficult, especially for eukaryotic species, and is often erroneous and incomplete. We used a proteogenomics strategy, taking advantage of the combination of proteomics datasets and bioinformatics tools, to identify novel protein coding-genes and splice isoforms, assign correct start sites, and validate predicted exons and genes. RESULTS: Our proteogenomics workflow, Peptimapper, was applied to the genome annotation of Ectocarpus sp., a key reference genome for both the brown algal lineage and stramenopiles. We generated proteomics data from various life cycle stages of Ectocarpus sp. strains and sub-cellular fractions using a shotgun approach. First, we directly generated peptide sequence tags (PSTs) from the proteomics data. Second, we mapped PSTs onto the translated genomic sequence. Closely located hits (i.e., PSTs locations on the genome) were then clustered to detect potential coding regions based on parameters optimized for the organism. Third, we evaluated each cluster and compared it to gene predictions from existing conventional genome annotation approaches. Finally, we integrated cluster locations into GFF files to use a genome viewer. We identified two potential novel genes, a ribosomal protein L22 and an aryl sulfotransferase and corrected the gene structure of a dihydrolipoamide acetyltransferase. We experimentally validated the results by RT-PCR and using transcriptomics data. CONCLUSIONS: Peptimapper is a complementary tool for the expert annotation of genomes. It is suitable for any organism and is distributed through a Docker image available on two public bioinformatics docker repositories: Docker Hub and BioShaDock. This workflow is also accessible through the Galaxy framework and for use by non-computer scientists at https://galaxy.protim.eu . Data are available via ProteomeXchange under identifier PXD010618.
Assuntos
Eucariotos/genética , Genoma , Anotação de Sequência Molecular , Proteogenômica/métodos , Software , Fluxo de Trabalho , Sequência de Aminoácidos , Códon/genética , Espectrometria de Massas , Peptídeos/química , Peptídeos/metabolismo , Reprodutibilidade dos TestesRESUMO
Higher plants, as autotrophic organisms, are effective sources of molecules. They hold great promise for metabolic engineering, but the behavior of plant metabolism at the network level is still incompletely described. Although structural models (stoichiometry matrices) and pathway databases are extremely useful, they cannot describe the complexity of the metabolic context, and new tools are required to visually represent integrated biocurated knowledge for use by both humans and computers. Here, we describe ChloroKB, a Web application (http://chlorokb.fr/) for visual exploration and analysis of the Arabidopsis (Arabidopsis thaliana) metabolic network in the chloroplast and related cellular pathways. The network was manually reconstructed through extensive biocuration to provide transparent traceability of experimental data. Proteins and metabolites were placed in their biological context (spatial distribution within cells, connectivity in the network, participation in supramolecular complexes, and regulatory interactions) using CellDesigner software. The network contains 1,147 reviewed proteins (559 localized exclusively in plastids, 68 in at least one additional compartment, and 520 outside the plastid), 122 proteins awaiting biochemical/genetic characterization, and 228 proteins for which genes have not yet been identified. The visual presentation is intuitive and browsing is fluid, providing instant access to the graphical representation of integrated processes and to a wealth of refined qualitative and quantitative data. ChloroKB will be a significant support for structural and quantitative kinetic modeling, for biological reasoning, when comparing novel data with established knowledge, for computer analyses, and for educational purposes. ChloroKB will be enhanced by continuous updates following contributions from plant researchers.
Assuntos
Cloroplastos/metabolismo , Internet , Bases de Conhecimento , Redes e Vias Metabólicas , Arabidopsis/metabolismo , Frações Subcelulares/metabolismoRESUMO
The present study is a contribution to the "neXt50 challenge", a coordinated effort across C-HPP teams to identify the 50 most tractable missing proteins (MPs) on each chromosome. We report the targeted search of 38 theoretically detectable MPs from chromosomes 2 and 14 in Triton X-100 soluble and insoluble sperm fractions from a total of 15 healthy donors. A targeted mass-spectrometry-based strategy consisting of the development of LC-PRM assays (with heavy labeled synthetic peptides) targeting 92 proteotypic peptides of the 38 selected MPs was used. Out of the 38 selected MPs, 12 were identified with two or more peptides and 3 with one peptide after extensive SDS-PAGE fractionation of the two samples and with overall low-intensity signals. The PRM data are available via ProteomeXchange in PASSEL (PASS01013). Further validation by immunohistochemistry on human testes sections and cytochemistry on sperm smears was performed for eight MPs with antibodies available from the Human Protein Atlas. Deep analysis of human sperm still allows the validation of MPs and therefore contributes to the C-HPP worldwide effort. We anticipate that our results will be of interest to the reproductive biology community because an in-depth analysis of these MPs may identify potential new candidates in the context of human idiopathic infertilities.
Assuntos
Proteoma/análise , Espermatozoides/química , Anticorpos , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 2/genética , Histocitoquímica , Humanos , Imuno-Histoquímica , Masculino , Octoxinol , Espectrometria de Massas em Tandem , Testículo/químicaRESUMO
Within the C-HPP, the Swiss and French teams are responsible for the annotation of proteins from chromosomes 2 and 14, respectively. neXtProt currently reports 1231 entries on chromosome 2 and 624 entries on chromosome 14; of these, 134 and 93 entries are still not experimentally validated and are thus considered as "missing proteins" (PE2-4), respectively. Among these entries, some may never be validated by conventional MS/MS approaches because of incompatible biochemical features. Others have already been validated but are still awaiting annotation. On the basis of information retrieved from the literature and from three of the main C-HPP resources (Human Protein Atlas, PeptideAtlas, and neXtProt), a subset of 40 theoretically detectable missing proteins (25 on chromosome 2 and 15 on chromosome 14) was defined for upcoming targeted studies in sperm samples. This list is proposed as a roadmap for the French and Swiss teams in the near future.
Assuntos
Cromossomos Humanos Par 14 , Cromossomos Humanos Par 2 , Proteoma/análise , Biologia Computacional/tendências , Mineração de Dados/tendências , Bases de Dados de Proteínas , França , Humanos , Masculino , Espermatozoides/química , Suíça , Espectrometria de Massas em Tandem/normasRESUMO
Every data-rich community research effort requires a clear plan for ensuring the quality of the data interpretation and comparability of analyses. To address this need within the Human Proteome Project (HPP) of the Human Proteome Organization (HUPO), we have developed through broad consultation a set of mass spectrometry data interpretation guidelines that should be applied to all HPP data contributions. For submission of manuscripts reporting HPP protein identification results, the guidelines are presented as a one-page checklist containing 15 essential points followed by two pages of expanded description of each. Here we present an overview of the guidelines and provide an in-depth description of each of the 15 elements to facilitate understanding of the intentions and rationale behind the guidelines, for both authors and reviewers. Broadly, these guidelines provide specific directions regarding how HPP data are to be submitted to mass spectrometry data repositories, how error analysis should be presented, and how detection of novel proteins should be supported with additional confirmatory evidence. These guidelines, developed by the HPP community, are presented to the broader scientific community for further discussion.
Assuntos
Guias como Assunto , Espectrometria de Massas/normas , Proteoma/química , Proteômica/organização & administração , Bases de Dados Factuais , Bases de Dados de Proteínas , Humanos , Proteômica/normasRESUMO
The Chromosome-Centric Human Proteome Project (C-HPP) aims to identify "missing" proteins in the neXtProt knowledgebase. We present an in-depth proteomics analysis of the human sperm proteome to identify testis-enriched missing proteins. Using protein extraction procedures and LC-MS/MS analysis, we detected 235 proteins (PE2-PE4) for which no previous evidence of protein expression was annotated. Through LC-MS/MS and LC-PRM analysis, data mining, and immunohistochemistry, we confirmed the expression of 206 missing proteins (PE2-PE4) in line with current HPP guidelines (version 2.0). Parallel reaction monitoring acquisition and sythetic heavy labeled peptides targeted 36 âªone-hit wonderâ« candidates selected based on prior peptide spectrum match assessment. 24 were validated with additional predicted and specifically targeted peptides. Evidence was found for 16 more missing proteins using immunohistochemistry on human testis sections. The expression pattern for some of these proteins was specific to the testis, and they could possibly be valuable markers with fertility assessment applications. Strong evidence was also found of four "uncertain" proteins (PE5); their status should be re-examined. We show how using a range of sample preparation techniques combined with MS-based analysis, expert knowledge, and complementary antibody-based techniques can produce data of interest to the community. All MS/MS data are available via ProteomeXchange under identifier PXD003947. In addition to contributing to the C-HPP, we hope these data will stimulate continued exploration of the sperm proteome.
Assuntos
Proteoma/análise , Espermatozoides/química , Cromatografia Líquida , Mineração de Dados , Bases de Dados de Proteínas , Humanos , Imuno-Histoquímica , Masculino , Proteômica/métodos , Espectrometria de Massas em Tandem , Testículo/químicaRESUMO
In the framework of the C-HPP, our Franco-Swiss consortium has adopted chromosomes 2 and 14, coding for a total of 382 missing proteins (proteins for which evidence is lacking at protein level). Over the last 4 years, the French proteomics infrastructure has collected high-quality data sets from 40 human samples, including a series of rarely studied cell lines, tissue types, and sample preparations. Here we described a step-by-step strategy based on the use of bioinformatics screening and subsequent mass spectrometry (MS)-based validation to identify what were up to now missing proteins in these data sets. Screening database search results (85,326 dat files) identified 58 of the missing proteins (36 on chromosome 2 and 22 on chromosome 14) by 83 unique peptides following the latest release of neXtProt (2014-09-19). PSMs corresponding to these peptides were thoroughly examined by applying two different MS-based criteria: peptide-level false discovery rate calculation and expert PSM quality assessment. Synthetic peptides were then produced and used to generate reference MS/MS spectra. A spectral similarity score was then calculated for each pair of reference-endogenous spectra and used as a third criterion for missing protein validation. Finally, LC-SRM assays were developed to target proteotypic peptides from four of the missing proteins detected in tissue/cell samples, which were still available and for which sample preparation could be reproduced. These LC-SRM assays unambiguously detected the endogenous unique peptide for three of the proteins. For two of these, identification was confirmed by additional proteotypic peptides. We concluded that of the initial set of 58 proteins detected by the bioinformatics screen, the consecutive MS-based validation criteria led to propose the identification of 13 of these proteins (8 on chromosome 2 and 5 on chromosome 14) that passed at least two of the three MS-based criteria. Thus, a rigorous step-by-step approach combining bioinformatics screening and MS-based validation assays is particularly suitable to obtain protein-level evidence for proteins previously considered as missing. All MS/MS data have been deposited in ProteomeXchange under identifier PXD002131.
Assuntos
Cromossomos Humanos Par 14 , Cromossomos Humanos Par 2 , Proteínas/genética , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Cromatografia Líquida , Humanos , Dados de Sequência Molecular , Proteínas/químicaRESUMO
Quantitative analyses and models are required to connect a plant's cellular organisation with its metabolism. However, quantitative data are often scattered over multiple studies, and finding such data and converting them into useful information is time-consuming. Consequently, there is a need to centralise the available data and to highlight the remaining knowledge gaps. Here, we present a step-by-step approach to manually extract quantitative data from various information sources, and to unify the data format. First, data from Arabidopsis leaf were collated, checked for consistency and correctness and curated by cross-checking sources. Second, quantitative data were combined by applying calculation rules. They were then integrated into a unique comprehensive, referenced, modifiable and reusable data compendium representing an Arabidopsis reference leaf. This atlas contains the metrics of the 15 cell types found in leaves at the cellular and subcellular levels.
RESUMO
Pseudomonas aeruginosa is a Gram-negative bacterium that can inhabit a wide variety of environments and infect different hosts. Human infections by this microbe are nowadays perceived as a major health problem due notably to its multiresistance. The present data set provides the first description of P. aeruginosa inner membrane proteome. To achieve this, we combined efficient separation of membranes from PAO1 reference strain using discontinuous sucrose gradient centrifugation and MS-based proteomic analysis. A core list of 991 nonredundant proteins was established and analyzed in terms of trans-membrane domains, signal peptide, and lipobox sequence prediction. Furthermore, functional insights into membrane-spanning and membrane-associated protein complexes have been explored. All mass spectrometry data have been deposited in the ProteomeXchange with identifier PXD000107.
Assuntos
Proteínas de Bactérias/análise , Proteínas de Membrana/análise , Proteoma/análise , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/citologia , Proteínas de Bactérias/química , Membrana Celular/química , Bases de Dados de Proteínas , Proteínas de Membrana/química , Proteômica/métodos , Espectrometria de Massas em TandemRESUMO
The cellular slime mold Dictyostelium discoideum is a soil-living eukaryote, which feeds on microorganisms engulfed by phagocytosis. Axenic laboratory strains have been produced that are able to use liquid growth medium internalized by macropinocytosis as the source of food. To better define the macropinocytosis process, we established the inventory of proteins associated with this pathway using mass spectrometry-based proteomics. Using a magnetic purification procedure and high-performance LC-MS/MS proteome analysis, a list of 2108 non-redundant proteins was established, of which 24% featured membrane-spanning domains. Bioinformatics analyses indicated that the most abundant proteins were linked to signaling, vesicular trafficking and the cytoskeleton. The present repertoire validates our purification method and paves the way for a future proteomics approach to study the dynamics of macropinocytosis.
Assuntos
Dictyostelium/metabolismo , Espectrometria de Massas/métodos , Pinocitose , Proteoma/análise , Proteômica/métodos , Western Blotting , Cromatografia Líquida/métodos , Biologia Computacional , Eletroforese em Gel de Poliacrilamida , Lisossomos/metabolismo , Imãs , Transporte Proteico , Proteoma/isolamento & purificação , Proteoma/metabolismo , Proteínas de Protozoários/análise , Proteínas de Protozoários/metabolismoRESUMO
With the increased simplicity of producing proteomics data, the bottleneck has now shifted to the functional analysis of large lists of proteins to translate this primary level of information into meaningful biological knowledge. Tools implementing such approach are a powerful way to gain biological insights related to their samples, provided that biologists/clinicians have access to computational solutions even when they have little programming experience or bioinformatics support. To achieve this goal, we designed ProteoRE (Proteomics Research Environment), a unified online research service that provides end-users with a set of tools to interpret their proteomics data in a collaborative and reproducible manner. ProteoRE is built upon the Galaxy framework, a workflow system allowing for data and analysis persistence, and providing user interfaces to facilitate the interaction with tools dedicated to the functional and the visual analysis of proteomics datasets. A set of tools relying on computational methods selected for their complementarity in terms of functional analysis was developed and made accessible via the ProteoRE web portal. In this chapter, a step-by-step protocol linking these tools is designed to perform a functional annotation and GO-based enrichment analyses applied to a set of differentially expressed proteins as a use case. Analytical practices, guidelines as well as tips related to this strategy are also provided. Tools, datasets, and results are freely available at http://www.proteore.org , allowing researchers to reuse them.