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1.
Cell ; 180(4): 729-748.e26, 2020 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-32059776

RESUMEN

We undertook a comprehensive proteogenomic characterization of 95 prospectively collected endometrial carcinomas, comprising 83 endometrioid and 12 serous tumors. This analysis revealed possible new consequences of perturbations to the p53 and Wnt/ß-catenin pathways, identified a potential role for circRNAs in the epithelial-mesenchymal transition, and provided new information about proteomic markers of clinical and genomic tumor subgroups, including relationships to known druggable pathways. An extensive genome-wide acetylation survey yielded insights into regulatory mechanisms linking Wnt signaling and histone acetylation. We also characterized aspects of the tumor immune landscape, including immunogenic alterations, neoantigens, common cancer/testis antigens, and the immune microenvironment, all of which can inform immunotherapy decisions. Collectively, our multi-omic analyses provide a valuable resource for researchers and clinicians, identify new molecular associations of potential mechanistic significance in the development of endometrial cancers, and suggest novel approaches for identifying potential therapeutic targets.


Asunto(s)
Carcinoma/genética , Neoplasias Endometriales/genética , Regulación Neoplásica de la Expresión Génica , Proteoma/genética , Transcriptoma , Acetilación , Animales , Antígenos de Neoplasias/genética , Carcinoma/inmunología , Carcinoma/patología , Neoplasias Endometriales/inmunología , Neoplasias Endometriales/patología , Transición Epitelial-Mesenquimal/genética , Retroalimentación Fisiológica , Femenino , Inestabilidad Genómica , Humanos , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Repeticiones de Microsatélite , Fosforilación , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Transducción de Señal
2.
Cell ; 179(4): 964-983.e31, 2019 10 31.
Artículo en Inglés | MEDLINE | ID: mdl-31675502

RESUMEN

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.


Asunto(s)
Carcinoma de Células Renales/genética , Proteínas de Neoplasias/genética , Proteogenómica , Transcriptoma/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/patología , Supervivencia sin Enfermedad , Exoma/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Genoma Humano/genética , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/inmunología , Fosforilación Oxidativa , Fosforilación/genética , Transducción de Señal/genética , Transcriptoma/inmunología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Secuenciación del Exoma
4.
J Proteome Res ; 23(1): 71-83, 2024 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-38112105

RESUMEN

Tyrosine sulfation in the Golgi of secreted and membrane proteins is an important post-translational modification (PTM). However, its labile nature has limited analysis by mass spectrometry (MS), a major reason why no sulfoproteome studies have been previously reported. Here, we show that a phosphoproteomics experimental workflow, which includes serial enrichment followed by high resolution, high mass accuracy MS, and tandem MS (MS/MS) analysis, enables sulfopeptide coenrichment and identification via accurate precursor ion mass shift open MSFragger database search. This approach, supported by manual validation, allows the confident identification of sulfotyrosine-containing peptides in the presence of high levels of phosphorylated peptides, thus enabling these two sterically and ionically similar isobaric PTMs to be distinguished and annotated in a single proteomic analysis. We applied this approach to isolated interphase and mitotic rat liver Golgi membranes and identified 67 tyrosine sulfopeptides, corresponding to 26 different proteins. This work discovered 23 new sulfoproteins with functions related to, for example, Ca2+-binding, glycan biosynthesis, and exocytosis. In addition, we report the first preliminary evidence for crosstalk between sulfation and phosphorylation in the Golgi, with implications for functional control.


Asunto(s)
Proteómica , Espectrometría de Masas en Tándem , Secuencia de Aminoácidos , Espectrometría de Masas en Tándem/métodos , Flujo de Trabajo , Péptidos/química , Tirosina/metabolismo , Procesamiento Proteico-Postraduccional
5.
Mol Cell Proteomics ; 20: 100018, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33568339

RESUMEN

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.


Asunto(s)
Péptidos/química , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Bases de Datos de Proteínas , Humanos , Ratones , Proteómica
6.
J Proteome Res ; 19(6): 2511-2515, 2020 06 05.
Artículo en Inglés | MEDLINE | ID: mdl-32338005

RESUMEN

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.


Asunto(s)
Proteómica , Espectrometría de Masas en Tándem , Bases de Datos de Proteínas , Péptidos , Procesamiento Proteico-Postraduccional
7.
Nat Methods ; 14(5): 513-520, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28394336

RESUMEN

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.


Asunto(s)
Biología Computacional/métodos , Fragmentos de Péptidos/química , Proteoma/química , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Algoritmos , Biología Computacional/instrumentación , Bases de Datos de Proteínas , Células HEK293 , Humanos , Procesamiento Proteico-Postraduccional , Proteómica/instrumentación
8.
J Proteome Res ; 18(2): 715-720, 2019 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-30523686

RESUMEN

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.


Asunto(s)
Procesamiento Proteico-Postraduccional , Proteómica/métodos , Programas Informáticos , Automatización , Bases de Datos de Proteínas , Peso Molecular
10.
Nat Commun ; 14(1): 4154, 2023 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-37438352

RESUMEN

Liquid chromatography (LC) coupled with data-independent acquisition (DIA) mass spectrometry (MS) has been increasingly used in quantitative proteomics studies. Here, we present a fast and sensitive approach for direct peptide identification from DIA data, MSFragger-DIA, which leverages the unmatched speed of the fragment ion indexing-based search engine MSFragger. Different from most existing methods, MSFragger-DIA conducts a database search of the DIA tandem mass (MS/MS) spectra prior to spectral feature detection and peak tracing across the LC dimension. To streamline the analysis of DIA data and enable easy reproducibility, we integrate MSFragger-DIA into the FragPipe computational platform for seamless support of peptide identification and spectral library building from DIA, data-dependent acquisition (DDA), or both data types combined. We compare MSFragger-DIA with other DIA tools, such as DIA-Umpire based workflow in FragPipe, Spectronaut, DIA-NN library-free, and MaxDIA. We demonstrate the fast, sensitive, and accurate performance of MSFragger-DIA across a variety of sample types and data acquisition schemes, including single-cell proteomics, phosphoproteomics, and large-scale tumor proteome profiling studies.


Asunto(s)
Proteómica , Espectrometría de Masas en Tándem , Reproducibilidad de los Resultados , Cromatografía Liquida , Bases de Datos Factuales
11.
Nat Commun ; 11(1): 4065, 2020 08 13.
Artículo en Inglés | MEDLINE | ID: mdl-32792501

RESUMEN

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.


Asunto(s)
Péptidos/química , Algoritmos , Animales , Bases de Datos de Proteínas , Humanos , Espectrometría de Masas , Proteómica/métodos
12.
Genome Biol ; 14(2): R13, 2013 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-23409723

RESUMEN

BACKGROUND: Protein-RNA interactions are integral components of nearly every aspect of biology, including regulation of gene expression, assembly of cellular architectures, and pathogenesis of human diseases. However, studies in the past few decades have only uncovered a small fraction of the vast landscape of the protein-RNA interactome in any organism, and even less is known about the dynamics of protein-RNA interactions under changing developmental and environmental conditions. RESULTS: Here, we describe the gPAR-CLIP (global photoactivatable-ribonucleoside-enhanced crosslinking and immunopurification) approach for capturing regions of the untranslated, polyadenylated transcriptome bound by RNA-binding proteins (RBPs) in budding yeast. We report over 13,000 RBP crosslinking sites in untranslated regions (UTRs) covering 72% of protein-coding transcripts encoded in the genome, confirming 3' UTRs as major sites for RBP interaction. Comparative genomic analyses reveal that RBP crosslinking sites are highly conserved, and RNA folding predictions indicate that secondary structural elements are constrained by protein binding and may serve as generalizable modes of RNA recognition. Finally, 38% of 3' UTR crosslinking sites show changes in RBP occupancy upon glucose or nitrogen deprivation, with major impacts on metabolic pathways as well as mitochondrial and ribosomal gene expression. CONCLUSIONS: Our study offers an unprecedented view of the pervasiveness and dynamics of protein-RNA interactions in vivo.


Asunto(s)
ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Transcriptoma , Regiones no Traducidas 3' , Sitios de Unión , Reactivos de Enlaces Cruzados , Unión Proteica , Pliegue del ARN , ARN Mensajero/química , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
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