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1.
Cell ; 179(4): 964-983.e31, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31675502

RESUMO

To elucidate the deregulated functional modules that drive clear cell renal cell carcinoma (ccRCC), we performed comprehensive genomic, epigenomic, transcriptomic, proteomic, and phosphoproteomic characterization of treatment-naive ccRCC and paired normal adjacent tissue samples. Genomic analyses identified a distinct molecular subgroup associated with genomic instability. Integration of proteogenomic measurements uniquely identified protein dysregulation of cellular mechanisms impacted by genomic alterations, including oxidative phosphorylation-related metabolism, protein translation processes, and phospho-signaling modules. To assess the degree of immune infiltration in individual tumors, we identified microenvironment cell signatures that delineated four immune-based ccRCC subtypes characterized by distinct cellular pathways. This study reports a large-scale proteogenomic analysis of ccRCC to discern the functional impact of genomic alterations and provides evidence for rational treatment selection stemming from ccRCC pathobiology.


Assuntos
Carcinoma de Células Renais/genética , Proteínas de Neoplasias/genética , Proteogenômica , Transcriptoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Intervalo Livre de Doença , Exoma/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Genoma Humano/genética , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/imunologia , Fosforilação Oxidativa , Fosforilação/genética , Transdução de Sinais/genética , Transcriptoma/imunologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Sequenciamento do Exoma
3.
Mol Cell Proteomics ; 20: 100018, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33568339

RESUMO

Open searching has proven to be an effective strategy for identifying both known and unknown modifications in shotgun proteomics experiments. Rather than being limited to a small set of user-specified modifications, open searches identify peptides with any mass shift that may correspond to a single modification or a combination of several modifications. Here we present PTM-Shepherd, a bioinformatics tool that automates characterization of post-translational modification profiles detected in open searches based on attributes, such as amino acid localization, fragmentation spectra similarity, retention time shifts, and relative modification rates. PTM-Shepherd can also perform multiexperiment comparisons for studying changes in modification profiles, e.g., in data generated in different laboratories or under different conditions. We demonstrate how PTM-Shepherd improves the analysis of data from formalin-fixed and paraffin-embedded samples, detects extreme underalkylation of cysteine in some data sets, discovers an artifactual modification introduced during peptide synthesis, and uncovers site-specific biases in sample preparation artifacts in a multicenter proteomics profiling study.


Assuntos
Peptídeos/química , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Bases de Dados de Proteínas , Humanos , Camundongos , Proteômica
4.
Mol Cell Proteomics ; 19(9): 1575-1585, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32616513

RESUMO

Ion mobility brings an additional dimension of separation to LC-MS, improving identification of peptides and proteins in complex mixtures. A recently introduced timsTOF mass spectrometer (Bruker) couples trapped ion mobility separation to TOF mass analysis. With the parallel accumulation serial fragmentation (PASEF) method, the timsTOF platform achieves promising results, yet analysis of the data generated on this platform represents a major bottleneck. Currently, MaxQuant and PEAKS are most used to analyze these data. However, because of the high complexity of timsTOF PASEF data, both require substantial time to perform even standard tryptic searches. Advanced searches (e.g. with many variable modifications, semi- or non-enzymatic searches, or open searches for post-translational modification discovery) are practically impossible. We have extended our fast peptide identification tool MSFragger to support timsTOF PASEF data, and developed a label-free quantification tool, IonQuant, for fast and accurate 4-D feature extraction and quantification. Using a HeLa data set published by Meier et al. (2018), we demonstrate that MSFragger identifies significantly (∼30%) more unique peptides than MaxQuant (1.6.10.43), and performs comparably or better than PEAKS X+ (∼10% more peptides). IonQuant outperforms both in terms of number of quantified proteins while maintaining good quantification precision and accuracy. Runtime tests show that MSFragger and IonQuant can fully process a typical two-hour PASEF run in under 70 min on a typical desktop (6 CPU cores, 32 GB RAM), significantly faster than other tools. Finally, through semi-enzymatic searching, we significantly increase the number of identified peptides. Within these semi-tryptic identifications, we report evidence of gas-phase fragmentation before MS/MS analysis.


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Mobilidade Iônica/métodos , Peptídeos/análise , Proteoma/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Algoritmos , Bases de Dados de Proteínas , Escherichia coli/metabolismo , Células HeLa , Humanos , Peptídeos/metabolismo , Filogenia , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Sensibilidade e Especificidade
5.
J Proteome Res ; 19(6): 2511-2515, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32338005

RESUMO

Shotgun proteomics using liquid chromatography coupled to mass spectrometry (LC-MS) is commonly used to identify peptides containing post-translational modifications. With the emergence of fast database search tools such as MSFragger, the approach of enlarging precursor mass tolerances during the search (termed "open search") has been increasingly used for comprehensive characterization of post-translational and chemical modifications of protein samples. However, not all mass shifts detected using the open search strategy represent true modifications, as artifacts exist from sources such as unaccounted missed cleavages or peptide co-fragmentation (chimeric MS/MS spectra). Here, we present Crystal-C, a computational tool that detects and removes such artifacts from open search results. Our analysis using Crystal-C shows that, in a typical shotgun proteomics data set, the number of such observations is relatively small. Nevertheless, removing these artifacts helps to simplify the interpretation of the mass shift histograms, which in turn should improve the ability of open search-based tools to detect potentially interesting mass shifts for follow-up investigation.


Assuntos
Proteômica , Espectrometria de Massas em Tandem , Bases de Dados de Proteínas , Peptídeos , Processamento de Proteína Pós-Traducional
6.
Nat Methods ; 14(5): 513-520, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28394336

RESUMO

There is a need to better understand and handle the 'dark matter' of proteomics-the vast diversity of post-translational and chemical modifications that are unaccounted in a typical mass spectrometry-based analysis and thus remain unidentified. We present a fragment-ion indexing method, and its implementation in peptide identification tool MSFragger, that enables a more than 100-fold improvement in speed over most existing proteome database search tools. Using several large proteomic data sets, we demonstrate how MSFragger empowers the open database search concept for comprehensive identification of peptides and all their modified forms, uncovering dramatic differences in modification rates across experimental samples and conditions. We further illustrate its utility using protein-RNA cross-linked peptide data and using affinity purification experiments where we observe, on average, a 300% increase in the number of identified spectra for enriched proteins. We also discuss the benefits of open searching for improved false discovery rate estimation in proteomics.


Assuntos
Biologia Computacional/métodos , Fragmentos de Peptídeos/química , Proteoma/química , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Algoritmos , Biologia Computacional/instrumentação , Bases de Dados de Proteínas , Células HEK293 , Humanos , Processamento de Proteína Pós-Traducional , Proteômica/instrumentação
7.
J Proteome Res ; 18(2): 715-720, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30523686

RESUMO

Routine identification of thousands of proteins in a single LC-MS experiment has long become the norm. With these vast amounts of data, more rigorous treatment of modified forms of peptides becomes possible. "Open search", a protein database search with a large precursor ion mass tolerance window, is becoming a popular method to evaluate possible sets of post-translational and chemical modifications in samples. The extraction of statistical information about the modification from peptide search results requires additional effort and data processing, such as recalibration of masses and accurate detection of precursors in MS1 signals. Here we present a software tool, DeltaMass, which performs kernel-density-based estimation of observed mass shifts and allows for the detection of poorly resolved mass deltas. The software also maps observed mass shifts to known modifications from public databases such as UniMod and augments them with additionally generated possible chemical changes to the molecule. Its interactive graphical interface provides an effective option for the visual interrogation of the data and the identification of potentially interesting mass shifts or unusual artifacts for subsequent analysis. However, the program can also be used in fully automated command-line mode to generate mass-shift peak lists as well.


Assuntos
Processamento de Proteína Pós-Traducional , Proteômica/métodos , Software , Automação , Bases de Dados de Proteínas , Peso Molecular
8.
Anal Chem ; 90(3): 2369-2375, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29278491

RESUMO

Top-down proteomics has emerged as a transformative method for the analysis of protein sequence and post-translational modifications (PTMs). Top-down experiments have historically been performed primarily on ultrahigh resolution mass spectrometers due to the complexity of spectra resulting from fragmentation of intact proteins, but recent advances in coupling ion mobility separations to faster, lower resolution mass analyzers now offer a viable alternative. However, software capable of interpreting the highly complex two-dimensional spectra that result from coupling ion mobility separation to top-down experiments is currently lacking. In this manuscript we present a software suite consisting of two programs, IMTBX ("IM Toolbox") and Grppr ("Grouper"), that enable fully automated processing of such data. We demonstrate the capabilities of this software suite by examining a series of intact proteins on a Waters Synapt G2 ion-mobility equipped mass spectrometer and compare the results to the manual and semiautomated data analysis procedures we have used previously.


Assuntos
Espectrometria de Massas/métodos , Proteômica/métodos , Software , Animais , Bovinos , Galinhas , Humanos , Proteínas/análise , Coelhos
10.
J Proteome Res ; 15(8): 2500-9, 2016 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-27306858

RESUMO

Mass spectrometry (MS) coupled to liquid chromatography (LC) is a commonly used technique in metabolomic and proteomic research. As the size and complexity of LC-MS-based experiments grow, it becomes increasingly more difficult to perform quality control of both raw data and processing results. In a practical setting, quality control steps for raw LC-MS data are often overlooked, and assessment of an experiment's success is based on some derived metrics such as "the number of identified compounds". The human brain interprets visual data much better than plain text, hence the saying "a picture is worth a thousand words". Here, we present the BatMass software package, which allows for performing quick quality control of raw LC-MS data through its fast visualization capabilities. It also serves as a testbed for developers of LC-MS data processing algorithms by providing a data access library for open mass spectrometry file formats and a means of visually mapping processing results back to the original data. We illustrate the utility of BatMass with several use cases of quality control and data exploration.


Assuntos
Gráficos por Computador , Espectrometria de Massas/métodos , Metabolômica/métodos , Proteômica/métodos , Software , Cromatografia Líquida , Controle de Qualidade
11.
Nat Commun ; 11(1): 4065, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792501

RESUMO

Identification of post-translationally or chemically modified peptides in mass spectrometry-based proteomics experiments is a crucial yet challenging task. We have recently introduced a fragment ion indexing method and the MSFragger search engine to empower an open search strategy for comprehensive analysis of modified peptides. However, this strategy does not consider fragment ions shifted by unknown modifications, preventing modification localization and limiting the sensitivity of the search. Here we present a localization-aware open search method, in which both modification-containing (shifted) and regular fragment ions are indexed and used in scoring. We also implement a fast mass calibration and optimization method, allowing optimization of the mass tolerances and other key search parameters. We demonstrate that MSFragger with mass calibration and localization-aware open search identifies modified peptides with significantly higher sensitivity and accuracy. Comparing MSFragger to other modification-focused tools (pFind3, MetaMorpheus, and TagGraph) shows that MSFragger remains an excellent option for fast, comprehensive, and sensitive searches for modified peptides in shotgun proteomics data.


Assuntos
Peptídeos/química , Algoritmos , Animais , Bases de Dados de Proteínas , Humanos , Espectrometria de Massas , Proteômica/métodos
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