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1.
Nat Commun ; 12(1): 4882, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385466

RESUMO

Genetic variants of the interferon lambda (IFNL) gene locus are strongly associated with spontaneous and IFN treatment-induced clearance of hepatitis C virus (HCV) infections. Individuals with the ancestral IFNL4-dG allele are not able to clear HCV in the acute phase and have more than a 90% probability to develop chronic hepatitis C (CHC). Paradoxically, the IFNL4-dG allele encodes a fully functional IFNλ4 protein with antiviral activity against HCV. Here we describe an effect of IFNλ4 on HCV antigen presentation. Only minor amounts of IFNλ4 are secreted, because the protein is largely retained in the endoplasmic reticulum (ER) where it induces ER stress. Stressed cells are significantly weaker activators of HCV specific CD8+ T cells than unstressed cells. This is not due to reduced MHC I surface presentation or extracellular IFNλ4 effects, since T cell responses are restored by exogenous loading of MHC with HCV antigens. Rather, IFNλ4 induced ER stress impairs HCV antigen processing and/or loading onto the MHC I complex. Our results provide a potential explanation for the IFNλ4-HCV paradox.


Assuntos
Apresentação do Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Hepacivirus/imunologia , Interleucinas/imunologia , Ativação Linfocitária/imunologia , Células A549 , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Linhagem Celular Tumoral , Regulação da Expressão Gênica/imunologia , Genótipo , Células Hep G2 , Hepacivirus/genética , Hepacivirus/fisiologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interleucinas/genética , Interleucinas/metabolismo
2.
J Proteome Res ; 20(7): 3758-3766, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34153189

RESUMO

Data-independent acquisition (DIA) is becoming a leading analysis method in biomedical mass spectrometry. The main advantages include greater reproducibility and sensitivity and a greater dynamic range compared with data-dependent acquisition (DDA). However, the data analysis is complex and often requires expert knowledge when dealing with large-scale data sets. Here we present DIAproteomics, a multifunctional, automated, high-throughput pipeline implemented in the Nextflow workflow management system that allows one to easily process proteomics and peptidomics DIA data sets on diverse compute infrastructures. The central components are well-established tools such as the OpenSwathWorkflow for the DIA spectral library search and PyProphet for the false discovery rate assessment. In addition, it provides options to generate spectral libraries from existing DDA data and to carry out the retention time and chromatogram alignment. The output includes annotated tables and diagnostic visualizations from the statistical postprocessing and computation of fold-changes across pairwise conditions, predefined in an experimental design. DIAproteomics is well documented open-source software and is available under a permissive license to the scientific community at https://www.openms.de/diaproteomics/.


Assuntos
Análise de Dados , Proteômica , Espectrometria de Massas , Reprodutibilidade dos Testes , Software
3.
Nat Commun ; 12(1): 3810, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155216

RESUMO

To a large extent functional diversity in cells is achieved by the expansion of molecular complexity beyond that of the coding genome. Various processes create multiple distinct but related proteins per coding gene - so-called proteoforms - that expand the functional capacity of a cell. Evaluating proteoforms from classical bottom-up proteomics datasets, where peptides instead of intact proteoforms are measured, has remained difficult. Here we present COPF, a tool for COrrelation-based functional ProteoForm assessment in bottom-up proteomics data. It leverages the concept of peptide correlation analysis to systematically assign peptides to co-varying proteoform groups. We show applications of COPF to protein complex co-fractionation data as well as to more typical protein abundance vs. sample data matrices, demonstrating the systematic detection of assembly- and tissue-specific proteoform groups, respectively, in either dataset. We envision that the presented approach lays the foundation for a systematic assessment of proteoforms and their functional implications directly from bottom-up proteomic datasets.


Assuntos
Isoformas de Proteínas/análise , Proteômica/métodos , Algoritmos , Animais , Benchmarking , Humanos , Camundongos , Peptídeos/análise , Peptídeos/metabolismo , Isoformas de Proteínas/metabolismo , Proteômica/normas , Espectrometria de Massas em Tandem , Fluxo de Trabalho
4.
Dev Cell ; 56(1): 111-124.e6, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33238149

RESUMO

To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the glutamic acids surrounding phosphosites significantly delay protein turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.


Assuntos
Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Fosfoproteínas/metabolismo , Proteólise , Proteoma/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Glutamatos/metabolismo , Humanos , Peptídeos/metabolismo , Peroxirredoxina VI/química , Peroxirredoxina VI/metabolismo , Fosfoproteínas/química , Fosforilação , Proteoma/genética , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Transdução de Sinais/genética
5.
Cell Syst ; 11(6): 589-607.e8, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33333029

RESUMO

Protein-protein interactions (PPIs) play critical functional and regulatory roles in cellular processes. They are essential for macromolecular complex formation, which in turn constitutes the basis for protein interaction networks that determine the functional state of a cell. We and others have previously shown that chromatographic fractionation of native protein complexes in combination with bottom-up mass spectrometric analysis of consecutive fractions supports the multiplexed characterization and detection of state-specific changes of protein complexes. In this study, we extend co-fractionation and mass spectrometric data analysis to perform quantitative, network-based studies of proteome organization, via the size-exclusion chromatography algorithmic toolkit (SECAT). This framework explicitly accounts for the dynamic nature and rewiring of protein complexes across multiple cell states and samples, thus, elucidating molecular mechanisms that are differentially implemented across different experimental settings. Systematic analysis of multiple datasets shows that SECAT represents a highly scalable and effective methodology to assess condition/state-specific protein-network state. A record of this paper's transparent peer review process is included in the Supplemental Information.


Assuntos
Espectrometria de Massas/métodos , Mapas de Interação de Proteínas/imunologia , Proteômica/métodos , Humanos
6.
J Proteome Res ; 19(10): 4163-4178, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-32966080

RESUMO

Proteoforms containing post-translational modifications (PTMs) represent a degree of functional diversity only harnessed through analytically precise simultaneous quantification of multiple PTMs. Here we present a method to accurately differentiate an unmodified peptide from its PTM-containing counterpart through data-independent acquisition-mass spectrometry, leveraging small precursor mass windows to physically separate modified peptidoforms from each other during MS2 acquisition. We utilize a lysine and arginine PTM-enriched peptide assay library and site localization algorithm to simultaneously localize and quantify seven PTMs including mono-, di-, and trimethylation, acetylation, and succinylation in addition to total protein quantification in a single MS run without the need to enrich experimental samples. To evaluate biological relevance, this method was applied to liver lysate from differentially methylated nonalcoholic steatohepatitis (NASH) mouse models. We report that altered methylation and acetylation together with total protein changes drive the novel hypothesis of a regulatory function of PTMs in protein synthesis and mRNA stability in NASH.


Assuntos
Hepatopatias , Lisina , Acetilação , Animais , Arginina , Lisina/metabolismo , Camundongos , Processamento de Proteína Pós-Traducional , Proteômica
7.
Nat Protoc ; 15(8): 2341-2386, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32690956

RESUMO

Most catalytic, structural and regulatory functions of the cell are carried out by functional modules, typically complexes containing or consisting of proteins. The composition and abundance of these complexes and the quantitative distribution of specific proteins across different modules are therefore of major significance in basic and translational biology. However, detection and quantification of protein complexes on a proteome-wide scale is technically challenging. We have recently extended the targeted proteomics rationale to the level of native protein complex analysis (complex-centric proteome profiling). The complex-centric workflow described herein consists of size exclusion chromatography (SEC) to fractionate native protein complexes, data-independent acquisition mass spectrometry to precisely quantify the proteins in each SEC fraction based on a set of proteotypic peptides and targeted, complex-centric analysis where prior information from generic protein interaction maps is used to detect and quantify protein complexes with high selectivity and statistical error control via the computational framework CCprofiler (https://github.com/CCprofiler/CCprofiler). Complex-centric proteome profiling captures most proteins in complex-assembled state and reveals their organization into hundreds of complexes and complex variants observable in a given cellular state. The protocol is applicable to cultured cells and can potentially also be adapted to primary tissue and does not require any genetic engineering of the respective sample sources. At present, it requires ~8 d of wet-laboratory work, 15 d of mass spectrometry measurement time and 7 d of computational analysis.


Assuntos
Cromatografia em Gel , Espectrometria de Massas , Proteínas/isolamento & purificação , Proteínas/metabolismo , Proteômica/métodos , Células HEK293 , Humanos
8.
Mol Cell ; 79(3): 504-520.e9, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32707033

RESUMO

Protein kinases are essential for signal transduction and control of most cellular processes, including metabolism, membrane transport, motility, and cell cycle. Despite the critical role of kinases in cells and their strong association with diseases, good coverage of their interactions is available for only a fraction of the 535 human kinases. Here, we present a comprehensive mass-spectrometry-based analysis of a human kinase interaction network covering more than 300 kinases. The interaction dataset is a high-quality resource with more than 5,000 previously unreported interactions. We extensively characterized the obtained network and were able to identify previously described, as well as predict new, kinase functional associations, including those of the less well-studied kinases PIM3 and protein O-mannose kinase (POMK). Importantly, the presented interaction map is a valuable resource for assisting biomedical studies. We uncover dozens of kinase-disease associations spanning from genetic disorders to complex diseases, including cancer.


Assuntos
Redes Reguladoras de Genes , Doenças Genéticas Inatas/genética , Neoplasias/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Regulação da Expressão Gênica , Ontologia Genética , Doenças Genéticas Inatas/enzimologia , Doenças Genéticas Inatas/patologia , Humanos , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular , Distrofias Musculares/enzimologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , Neoplasias/enzimologia , Neoplasias/patologia , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Mapeamento de Interação de Proteínas/métodos , Proteínas Quinases/química , Proteínas Quinases/classificação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais
9.
Mol Syst Biol ; 16(3): e9170, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32175694

RESUMO

Profiling of biological relationships between different molecular layers dissects regulatory mechanisms that ultimately determine cellular function. To thoroughly assess the role of protein post-translational turnover, we devised a strategy combining pulse stable isotope-labeled amino acids in cells (pSILAC), data-independent acquisition mass spectrometry (DIA-MS), and a novel data analysis framework that resolves protein degradation rate on the level of mRNA alternative splicing isoforms and isoform groups. We demonstrated our approach by the genome-wide correlation analysis between mRNA amounts and protein degradation across different strains of HeLa cells that harbor a high grade of gene dosage variation. The dataset revealed that specific biological processes, cellular organelles, spatial compartments of organelles, and individual protein isoforms of the same genes could have distinctive degradation rate. The protein degradation diversity thus dissects the corresponding buffering or concerting protein turnover control across cancer cell lines. The data further indicate that specific mRNA splicing events such as intron retention significantly impact the protein abundance levels. Our findings support the tight association between transcriptome variability and proteostasis and provide a methodological foundation for studying functional protein degradation.


Assuntos
Isoformas de Proteínas/análise , Proteínas/análise , Isoformas de RNA/metabolismo , RNA Mensageiro/metabolismo , Processamento Alternativo , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Marcação por Isótopo/métodos , Espectrometria de Massas , Isoformas de Proteínas/metabolismo , Proteínas/metabolismo , Proteólise , Proteômica/métodos , Isoformas de RNA/genética , RNA Mensageiro/genética , Fluxo de Trabalho
10.
Cell Syst ; 10(2): 133-155.e6, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32027860

RESUMO

Living systems integrate biochemical reactions that determine the functional state of each cell. Reactions are primarily mediated by proteins. In proteomic studies, these have been treated as independent entities, disregarding their higher-level organization into complexes that affects their activity and/or function and is thus of great interest for biological research. Here, we describe the implementation of an integrated technique to quantify cell-state-specific changes in the physical arrangement of protein complexes concurrently for thousands of proteins and hundreds of complexes. Applying this technique to a comparison of human cells in interphase and mitosis, we provide a systematic overview of mitotic proteome reorganization. The results recall key hallmarks of mitotic complex remodeling and suggest a model of nuclear pore complex disassembly, which we validate by orthogonal methods. To support the interpretation of quantitative SEC-SWATH-MS datasets, we extend the software CCprofiler and provide an interactive exploration tool, SECexplorer-cc.


Assuntos
Mitose/genética , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Humanos
11.
12.
J Am Soc Mass Spectrom ; 30(8): 1396-1405, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31147889

RESUMO

Due to the technical advances of mass spectrometers, particularly increased scanning speed and higher MS/MS resolution, the use of data-independent acquisition mass spectrometry (DIA-MS) became more popular, which enables high reproducibility in both proteomic identification and quantification. The current DIA-MS methods normally cover a wide mass range, with the aim to target and identify as many peptides and proteins as possible and therefore frequently generate MS/MS spectra of high complexity. In this report, we assessed the performance and benefits of using small windows with, e.g., 5-m/z width across the peptide elution time. We further devised a new DIA method named RTwinDIA that schedules the small isolation windows in different retention time blocks, taking advantage of the fact that larger peptides are normally eluting later in reversed phase chromatography. We assessed the direct proteomic identification by using shotgun database searching tools such as MaxQuant and pFind, and also Spectronaut with an external comprehensive spectral library of human proteins. We conclude that algorithms like pFind have potential in directly analyzing DIA data acquired with small windows, and that the instrumental time and DIA cycle time, if prioritized to be spent on small windows rather than on covering a broad mass range by large windows, will improve the direct proteome coverage for new biological samples and increase the quantitative precision. These results further provide perspectives for the future convergence between DDA and DIA on faster MS analyzers.


Assuntos
Proteínas/análise , Proteômica/métodos , Linhagem Celular Tumoral , Cromatografia de Fase Reversa , Humanos , Espectrometria de Massas/métodos , Peptídeos/análise , Software
13.
Mol Syst Biol ; 15(1): e8438, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30642884

RESUMO

Proteins are major effectors and regulators of biological processes that can elicit multiple functions depending on their interaction with other proteins. The organization of proteins into macromolecular complexes and their quantitative distribution across these complexes is, therefore, of great biological and clinical significance. In this paper, we describe an integrated experimental and computational technique to quantify hundreds of protein complexes in a single operation. The method consists of size exclusion chromatography (SEC) to fractionate native protein complexes, SWATH/DIA mass spectrometry to precisely quantify the proteins in each SEC fraction, and the computational framework CCprofiler to detect and quantify protein complexes by error-controlled, complex-centric analysis using prior information from generic protein interaction maps. Our analysis of the HEK293 cell line proteome delineates 462 complexes composed of 2,127 protein subunits. The technique identifies novel sub-complexes and assembly intermediates of central regulatory complexes while assessing the quantitative subunit distribution across them. We make the toolset CCprofiler freely accessible and provide a web platform, SECexplorer, for custom exploration of the HEK293 proteome modularity.


Assuntos
Cromatografia em Gel/métodos , Espectrometria de Massas/métodos , Complexos Multiproteicos/análise , Proteoma/análise , Proteômica/métodos , Algoritmos , Biologia Computacional/métodos , Células HEK293 , Humanos , Complexos Multiproteicos/metabolismo , Mapas de Interação de Proteínas , Proteoma/metabolismo
14.
Mol Syst Biol ; 14(8): e8126, 2018 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-30104418

RESUMO

Many research questions in fields such as personalized medicine, drug screens or systems biology depend on obtaining consistent and quantitatively accurate proteomics data from many samples. SWATH-MS is a specific variant of data-independent acquisition (DIA) methods and is emerging as a technology that combines deep proteome coverage capabilities with quantitative consistency and accuracy. In a SWATH-MS measurement, all ionized peptides of a given sample that fall within a specified mass range are fragmented in a systematic and unbiased fashion using rather large precursor isolation windows. To analyse SWATH-MS data, a strategy based on peptide-centric scoring has been established, which typically requires prior knowledge about the chromatographic and mass spectrometric behaviour of peptides of interest in the form of spectral libraries and peptide query parameters. This tutorial provides guidelines on how to set up and plan a SWATH-MS experiment, how to perform the mass spectrometric measurement and how to analyse SWATH-MS data using peptide-centric scoring. Furthermore, concepts on how to improve SWATH-MS data acquisition, potential trade-offs of parameter settings and alternative data analysis strategies are discussed.


Assuntos
Cromatografia Líquida , Peptídeos/genética , Proteômica/métodos , Espectrometria de Massas em Tandem , Proteoma , Proteômica/tendências , Software , Biologia de Sistemas/tendências
15.
Nat Commun ; 8(1): 291, 2017 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-28827567

RESUMO

Quantitative proteomics employing mass spectrometry is an indispensable tool in life science research. Targeted proteomics has emerged as a powerful approach for reproducible quantification but is limited in the number of proteins quantified. SWATH-mass spectrometry consists of data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, and selectivity) of targeted proteomics at large scale. While previous SWATH-mass spectrometry studies have shown high intra-lab reproducibility, this has not been evaluated between labs. In this multi-laboratory evaluation study including 11 sites worldwide, we demonstrate that using SWATH-mass spectrometry data acquisition we can consistently detect and reproducibly quantify >4000 proteins from HEK293 cells. Using synthetic peptide dilution series, we show that the sensitivity, dynamic range and reproducibility established with SWATH-mass spectrometry are uniformly achieved. This study demonstrates that the acquisition of reproducible quantitative proteomics data by multiple labs is achievable, and broadly serves to increase confidence in SWATH-mass spectrometry data acquisition as a reproducible method for large-scale protein quantification.SWATH-mass spectrometry consists of a data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics on the scale of thousands of proteins. Here, using data generated by eleven groups worldwide, the authors show that SWATH-MS is capable of generating highly reproducible data across different laboratories.


Assuntos
Ensaio de Proficiência Laboratorial/métodos , Espectrometria de Massas/métodos , Proteoma/metabolismo , Proteômica/métodos , Células HEK293 , Humanos , Laboratórios/normas , Laboratórios/estatística & dados numéricos , Reprodutibilidade dos Testes
16.
Nat Methods ; 14(9): 921-927, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28825704

RESUMO

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the main method for high-throughput identification and quantification of peptides and inferred proteins. Within this field, data-independent acquisition (DIA) combined with peptide-centric scoring, as exemplified by the technique SWATH-MS, has emerged as a scalable method to achieve deep and consistent proteome coverage across large-scale data sets. We demonstrate that statistical concepts developed for discovery proteomics based on spectrum-centric scoring can be adapted to large-scale DIA experiments that have been analyzed with peptide-centric scoring strategies, and we provide guidance on their application. We show that optimal tradeoffs between sensitivity and specificity require careful considerations of the relationship between proteins in the samples and proteins represented in the spectral library. We propose the application of a global analyte constraint to prevent the accumulation of false positives across large-scale data sets. Furthermore, to increase the quality and reproducibility of published proteomic results, well-established confidence criteria should be reported for the detected peptide queries, peptides and inferred proteins.


Assuntos
Interpretação Estatística de Dados , Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas/métodos , Mapeamento de Peptídeos/métodos , Proteínas/química , Análise de Sequência de Proteína/métodos , Simulação por Computador , Modelos Estatísticos , Proteínas/análise , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
17.
Nat Biotechnol ; 35(8): 781-788, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28604659

RESUMO

Consistent detection and quantification of protein post-translational modifications (PTMs) across sample cohorts is a prerequisite for functional analysis of biological processes. Data-independent acquisition (DIA) is a bottom-up mass spectrometry approach that provides complete information on precursor and fragment ions. However, owing to the convoluted structure of DIA data sets, confident, systematic identification and quantification of peptidoforms has remained challenging. Here, we present inference of peptidoforms (IPF), a fully automated algorithm that uses spectral libraries to query, validate and quantify peptidoforms in DIA data sets. The method was developed on data acquired by the DIA method SWATH-MS and benchmarked using a synthetic phosphopeptide reference data set and phosphopeptide-enriched samples. IPF reduced false site-localization by more than sevenfold compared with previous approaches, while recovering 85.4% of the true signals. Using IPF, we quantified peptidoforms in DIA data acquired from >200 samples of blood plasma of a human twin cohort and assessed the contribution of heritable, environmental and longitudinal effects on their PTMs.


Assuntos
Espectrometria de Massas/métodos , Peptídeos/sangue , Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Algoritmos , Apolipoproteína A-I/química , Humanos , Fosfopeptídeos/sangue , Fosfopeptídeos/química , Gêmeos
18.
Nat Protoc ; 12(7): 1289-1294, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28569762

RESUMO

In this Perspective, we discuss developments in mass-spectrometry-based proteomic technology over the past decade from the viewpoint of our laboratory. We also reflect on existing challenges and limitations, and explore the current and future roles of quantitative proteomics in molecular systems biology, clinical research and personalized medicine.


Assuntos
Espectrometria de Massas/métodos , Proteômica/métodos , Técnicas de Laboratório Clínico/métodos , Humanos , Proteômica/tendências , Biologia de Sistemas/métodos
19.
Nat Biotechnol ; 34(11): 1130-1136, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27701404

RESUMO

Consistent and accurate quantification of proteins by mass spectrometry (MS)-based proteomics depends on the performance of instruments, acquisition methods and data analysis software. In collaboration with the software developers, we evaluated OpenSWATH, SWATH 2.0, Skyline, Spectronaut and DIA-Umpire, five of the most widely used software methods for processing data from sequential window acquisition of all theoretical fragment-ion spectra (SWATH)-MS, which uses data-independent acquisition (DIA) for label-free protein quantification. We analyzed high-complexity test data sets from hybrid proteome samples of defined quantitative composition acquired on two different MS instruments using different SWATH isolation-window setups. For consistent evaluation, we developed LFQbench, an R package, to calculate metrics of precision and accuracy in label-free quantitative MS and report the identification performance, robustness and specificity of each software tool. Our reference data sets enabled developers to improve their software tools. After optimization, all tools provided highly convergent identification and reliable quantification performance, underscoring their robustness for label-free quantitative proteomics.


Assuntos
Benchmarking/métodos , Benchmarking/normas , Espectrometria de Massas/normas , Proteoma/química , Software/classificação , Software/normas , Algoritmos , Internacionalidade , Proteoma/análise , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e Rotulagem
20.
Nat Methods ; 13(9): 741-8, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27575624

RESUMO

High-resolution mass spectrometry (MS) has become an important tool in the life sciences, contributing to the diagnosis and understanding of human diseases, elucidating biomolecular structural information and characterizing cellular signaling networks. However, the rapid growth in the volume and complexity of MS data makes transparent, accurate and reproducible analysis difficult. We present OpenMS 2.0 (http://www.openms.de), a robust, open-source, cross-platform software specifically designed for the flexible and reproducible analysis of high-throughput MS data. The extensible OpenMS software implements common mass spectrometric data processing tasks through a well-defined application programming interface in C++ and Python and through standardized open data formats. OpenMS additionally provides a set of 185 tools and ready-made workflows for common mass spectrometric data processing tasks, which enable users to perform complex quantitative mass spectrometric analyses with ease.


Assuntos
Biologia Computacional/métodos , Processamento Eletrônico de Dados , Espectrometria de Massas/métodos , Proteômica/métodos , Software , Envelhecimento/sangue , Proteínas Sanguíneas/química , Humanos , Anotação de Sequência Molecular , Proteogenômica/métodos , Fluxo de Trabalho
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