ABSTRACT
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.
Subject(s)
Carcinoma, Renal Cell/genetics , Neoplasm Proteins/genetics , Proteogenomics , Transcriptome/genetics , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/genetics , Biomarkers, Tumor/immunology , Carcinoma, Renal Cell/immunology , Carcinoma, Renal Cell/pathology , Disease-Free Survival , Exome/genetics , Female , Gene Expression Regulation, Neoplastic/genetics , Genome, Human/genetics , Humans , Male , Middle Aged , Neoplasm Proteins/immunology , Oxidative Phosphorylation , Phosphorylation/genetics , Signal Transduction/genetics , Transcriptome/immunology , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology , Exome SequencingABSTRACT
Posttranslational modifications of proteins play essential roles in defining and regulating the functions of the proteins they decorate, making identification of these modifications critical to understanding biology and disease. Methods for enriching and analyzing a wide variety of biological and chemical modifications of proteins have been developed using mass spectrometry-based proteomics, largely relying on traditional database search methods to identify the resulting mass spectra of modified peptides. These database search methods treat modifications as static attachments of a mass to particular position in the peptide sequence, but many modifications undergo fragmentation in tandem mass spectrometry experiments alongside, or instead of, the peptide backbone. While this fragmentation can confound traditional search methods, it also offers unique opportunities for improved searches that incorporate modification-specific fragment ions. Here, we present a new labile mode in the MSFragger search engine that provides the flexibility to tailor modification-centric searches to the fragmentation observed. We show that labile mode can dramatically improve spectrum identification rates of phosphopeptides, RNA-crosslinked peptides, and ADP-ribosylated peptides. Each of these modifications presents distinct fragmentation characteristics, showcasing the flexibility of MSFragger labile mode to improve search for a wide variety of biological and chemical modifications.
Subject(s)
Protein Processing, Post-Translational , Proteomics , Proteomics/methods , Proteins/metabolism , Tandem Mass Spectrometry/methods , Phosphopeptides/metabolism , Databases, ProteinABSTRACT
Recent advances in methods for enrichment and mass spectrometric analysis of intact glycopeptides have produced large-scale glycoproteomics datasets, but interpreting these data remains challenging. We present MSFragger-Glyco, a glycoproteomics mode of the MSFragger search engine, for fast and sensitive identification of N- and O-linked glycopeptides and open glycan searches. Reanalysis of recent N-glycoproteomics data resulted in annotation of 80% more glycopeptide spectrum matches (glycoPSMs) than previously reported. In published O-glycoproteomics data, our method more than doubled the number of glycoPSMs annotated when searching the same glycans as the original search, and yielded 4- to 6-fold increases when expanding searches to include additional glycan compositions and other modifications. Expanded searches also revealed many sulfated and complex glycans that remained hidden to the original search. With greatly improved spectral annotation, coupled with the speed of index-based scoring, MSFragger-Glyco makes it possible to comprehensively interrogate glycoproteomics data and illuminate the many roles of glycosylation.
Subject(s)
Glycopeptides , Proteomics/methods , Search Engine , Tandem Mass Spectrometry , Databases, Protein , Glycopeptides/analysis , Glycopeptides/chemistry , Glycosylation , Proteomics/instrumentationABSTRACT
MassIVE.quant is a repository infrastructure and data resource for reproducible quantitative mass spectrometry-based proteomics, which is compatible with all mass spectrometry data acquisition types and computational analysis tools. A branch structure enables MassIVE.quant to systematically store raw experimental data, metadata of the experimental design, scripts of the quantitative analysis workflow, intermediate input and output files, as well as alternative reanalyses of the same dataset.
Subject(s)
Databases, Protein , Mass Spectrometry , Proteomics , Algorithms , Fungal Proteins/chemistry , Reproducibility of Results , Saccharomyces cerevisiae/metabolism , SoftwareABSTRACT
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 (â¼30%) more unique peptides than MaxQuant (1.6.10.43), and performs comparably or better than PEAKS X+ (â¼10% 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.
Subject(s)
Chromatography, Liquid/methods , Ion Mobility Spectrometry/methods , Peptides/analysis , Proteome/metabolism , Proteomics/methods , Tandem Mass Spectrometry/methods , Algorithms , Databases, Protein , Escherichia coli/metabolism , HeLa Cells , Humans , Peptides/metabolism , Phylogeny , Protein Processing, Post-Translational , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/metabolism , Sensitivity and SpecificityABSTRACT
Deisotoping, or the process of removing peaks in a mass spectrum resulting from the incorporation of naturally occurring heavy isotopes, has long been used to reduce complexity and improve the effectiveness of spectral annotation methods in proteomics. We have previously described MSFragger, an ultrafast search engine for proteomics, that did not utilize deisotoping in processing input spectra. Here, we present a new, high-speed parallelized deisotoping algorithm, based on elements of several existing methods, that we have incorporated into the MSFragger search engine. Applying deisotoping with MSFragger reveals substantial improvements to database search speed and performance, particularly for complex methods like open or nonspecific searches. Finally, we evaluate our deisotoping method on data from several instrument types and vendors, revealing a wide range in performance and offering an updated perspective on deisotoping in the modern proteomics environment.
Subject(s)
Algorithms , Databases, Protein , Search Engine , Mass Spectrometry , Proteomics , SoftwareABSTRACT
Methodologies that facilitate high-throughput proteomic analysis are a key step toward moving proteome investigations into clinical translation. Data independent acquisition (DIA) has potential as a high-throughput analytical method due to the reduced time needed for sample analysis, as well as its highly quantitative accuracy. However, a limiting feature of DIA methods is the sensitivity of detection of low abundant proteins and depth of coverage, which other mass spectrometry approaches address by two-dimensional fractionation (2D) to reduce sample complexity during data acquisition. In this study, we developed a 2D-DIA method intended for rapid- and deeper-proteome analysis compared to conventional 1D-DIA analysis. First, we characterized 96 individual fractions obtained from the protein standard, NCI-7, using a data-dependent approach (DDA), identifying a total of 151,366 unique peptides from 11,273 protein groups. We observed that the majority of the proteins can be identified from just a few selected fractions. By performing an optimization analysis, we identified six fractions with high peptide number and uniqueness that can account for 80% of the proteins identified in the entire experiment. These selected fractions were combined into a single sample which was then subjected to DIA (referred to as 2D-DIA) quantitative analysis. Furthermore, improved DIA quantification was achieved using a hybrid spectral library, obtained by combining peptides identified from DDA data with peptides identified directly from the DIA runs with the help of DIA-Umpire. The optimized 2D-DIA method allowed for improved identification and quantification of low abundant proteins compared to conventional unfractionated DIA analysis (1D-DIA). We then applied the 2D-DIA method to profile the proteomes of two breast cancer patient-derived xenograft (PDX) models, quantifying 6,217 and 6,167 unique proteins in basal- and luminal- tumors, respectively. Overall, this study demonstrates the potential of high-throughput quantitative proteomics using a novel 2D-DIA method.
Subject(s)
Peptides/analysis , Proteins/analysis , Proteomics , Humans , Mass SpectrometryABSTRACT
We describe an improved version of the data-independent acquisition (DIA) computational analysis tool DIA-Umpire, and show that it enables highly sensitive, untargeted, and direct (spectral library-free) analysis of DIA data obtained using the Orbitrap family of mass spectrometers. DIA-Umpire v2 implements an improved feature detection algorithm with two additional filters based on the isotope pattern and fractional peptide mass analysis. The targeted re-extraction step of DIA-Umpire is updated with an improved scoring function and a more robust, semiparametric mixture modeling of the resulting scores for computing posterior probabilities of correct peptide identification in a targeted setting. Using two publicly available Q Exactive DIA datasets generated using HEK-293 cells and human liver microtissues, we demonstrate that DIA-Umpire can identify similar number of peptide ions, but with better identification reproducibility between replicates and samples, as with conventional data-dependent acquisition. We further demonstrate the utility of DIA-Umpire using a series of Orbitrap Fusion DIA experiments with HeLa cell lysates profiled using conventional data-dependent acquisition and using DIA with different isolation window widths.
Subject(s)
Computational Biology/methods , Mass Spectrometry/methods , Proteomics/methods , HEK293 Cells , HeLa Cells , HumansABSTRACT
Data-independent acquisition (DIA) has become a widely used strategy for peptide and protein quantification in mass spectrometry-based proteomics studies. The integration of ion mobility separation into DIA analysis, such as the diaPASEF technology available on Bruker's timsTOF platform, further improves the quantification accuracy and protein depth achievable using DIA. We introduce diaTracer, a new spectrum-centric computational tool optimized for diaPASEF data. diaTracer performs three-dimensional (m/z, retention time, ion mobility) peak tracing and feature detection to generate precursor-resolved "pseudo-MS/MS" spectra, facilitating direct ("spectral-library free") peptide identification and quantification from diaPASEF data. diaTracer is available as a stand-alone tool and is fully integrated into the widely used FragPipe computational platform. We demonstrate the performance of diaTracer and FragPipe using diaPASEF data from cerebrospinal fluid (CSF) and plasma samples, data from phosphoproteomics and HLA immunopeptidomics experiments, and low-input data from a spatial proteomics study. We also show that diaTracer enables unrestricted identification of post-translational modifications from diaPASEF data using open/mass offset searches.
ABSTRACT
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.
Subject(s)
Proteomics , Tandem Mass Spectrometry , Reproducibility of Results , Chromatography, Liquid , Databases, FactualABSTRACT
Peptide identification in liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments relies on computational algorithms for matching acquired MS/MS spectra against sequences of candidate peptides using database search tools, such as MSFragger. Here, we present a new tool, MSBooster, for rescoring peptide-to-spectrum matches using additional features incorporating deep learning-based predictions of peptide properties, such as LC retention time, ion mobility, and MS/MS spectra. We demonstrate the utility of MSBooster, in tandem with MSFragger and Percolator, in several different workflows, including nonspecific searches (immunopeptidomics), direct identification of peptides from data independent acquisition data, single-cell proteomics, and data generated on an ion mobility separation-enabled timsTOF MS platform. MSBooster is fast, robust, and fully integrated into the widely used FragPipe computational platform.
Subject(s)
Deep Learning , Tandem Mass Spectrometry , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Peptides/chemistry , Algorithms , Databases, ProteinABSTRACT
Post-translational modification (PTM) of antigens provides an additional source of specificities targeted by immune responses to tumors or pathogens, but identifying antigen PTMs and assessing their role in shaping the immunopeptidome is challenging. Here we describe the Protein Modification Integrated Search Engine (PROMISE), an antigen discovery pipeline that enables the analysis of 29 different PTM combinations from multiple clinical cohorts and cell lines. We expanded the antigen landscape, uncovering human leukocyte antigen class I binding motifs defined by specific PTMs with haplotype-specific binding preferences and revealing disease-specific modified targets, including thousands of new cancer-specific antigens that can be shared between patients and across cancer types. Furthermore, we uncovered a subset of modified peptides that are specific to cancer tissue and driven by post-translational changes that occurred in the tumor proteome. Our findings highlight principles of PTM-driven antigenicity, which may have broad implications for T cell-mediated therapies in cancer and beyond.
Subject(s)
Neoplasms , Protein Processing, Post-Translational , Humans , Protein Processing, Post-Translational/genetics , Peptides/genetics , Antigens , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Neoplasms/geneticsABSTRACT
Clear cell renal cell carcinomas (ccRCCs) represent â¼75% of RCC cases and account for most RCC-associated deaths. Inter- and intratumoral heterogeneity (ITH) results in varying prognosis and treatment outcomes. To obtain the most comprehensive profile of ccRCC, we perform integrative histopathologic, proteogenomic, and metabolomic analyses on 305 ccRCC tumor segments and 166 paired adjacent normal tissues from 213 cases. Combining histologic and molecular profiles reveals ITH in 90% of ccRCCs, with 50% demonstrating immune signature heterogeneity. High tumor grade, along with BAP1 mutation, genome instability, increased hypermethylation, and a specific protein glycosylation signature define a high-risk disease subset, where UCHL1 expression displays prognostic value. Single-nuclei RNA sequencing of the adverse sarcomatoid and rhabdoid phenotypes uncover gene signatures and potential insights into tumor evolution. In vitro cell line studies confirm the potential of inhibiting identified phosphoproteome targets. This study molecularly stratifies aggressive histopathologic subtypes that may inform more effective treatment strategies.
Subject(s)
Carcinoma, Renal Cell , Kidney Neoplasms , Proteogenomics , Humans , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Treatment Outcome , Prognosis , Biomarkers, Tumor/geneticsABSTRACT
The dia-PASEF technology uses ion mobility separation to reduce signal interferences and increase sensitivity in proteomic experiments. Here we present a two-dimensional peak-picking algorithm and generation of optimized spectral libraries, as well as take advantage of neural network-based processing of dia-PASEF data. Our computational platform boosts proteomic depth by up to 83% compared to previous work, and is specifically beneficial for fast proteomic experiments and those with low sample amounts. It quantifies over 5300 proteins in single injections recorded at 200 samples per day throughput using Evosep One chromatography system on a timsTOF Pro mass spectrometer and almost 9000 proteins in single injections recorded with a 93-min nanoflow gradient on timsTOF Pro 2, from 200 ng of HeLa peptides. A user-friendly implementation is provided through the incorporation of the algorithms in the DIA-NN software and by the FragPipe workflow for spectral library generation.
Subject(s)
Proteome , Proteomics , Data Analysis , Humans , Mass Spectrometry/methods , Peptides/analysis , Proteome/analysis , Proteomics/methodsABSTRACT
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.