Analysis of DIA proteomics data using MSFragger-DIA and FragPipe computational platform

Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)

Published: July 12, 2023

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 fragment ion indexing-based search engine MSFragger. Different most existing methods, MSFragger-DIA conducts database tandem (MS/MS) spectra prior to spectral feature detection peak tracing across LC dimension. To streamline analysis data enable easy reproducibility, integrate into FragPipe computational platform seamless support library building DIA, data-dependent (DDA), or both types combined. We compare other tools, such as DIA-Umpire based workflow FragPipe, Spectronaut, DIA-NN library-free, MaxDIA. demonstrate fast, sensitive, accurate performance variety sample schemes, including single-cell proteomics, phosphoproteomics, large-scale tumor proteome profiling

Language: Английский

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Acquisition and Analysis of DIA-Based Proteomic Data: A Comprehensive Survey in 2023

Molecular & Cellular Proteomics, Journal Year: 2024, Volume and Issue: 23(2), P. 100712 - 100712

Published: Jan. 4, 2024

Data-independent acquisition (DIA) mass spectrometry (MS) has emerged as a powerful technology for high-throughput, accurate and reproducible quantitative proteomics. This review provides comprehensive overview of recent advances in both the experimental computational methods DIA proteomics, from data schemes to analysis strategies software tools. are categorized based on design precursor isolation windows, highlighting wide-window, overlapping-window, narrow-window, scanning quadrupole-based, parallel accumulation-serial fragmentation (PASEF)-enhanced methods. For analysis, major classified into spectrum reconstruction, sequence-based search, library-based de novo sequencing sequencing-independent approaches. A wide array tools implementing these reviewed, with details their overall workflows scoring approaches at different steps. The generation optimization spectral libraries, which critical resources also discussed. Publicly available benchmark datasets covering global proteomics phosphoproteomics summarized facilitate performance evaluation various workflows. Continued synergistic developments versatile components expected further enhance power DIA-based

Language: Английский

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Immunopeptidomics-based identification of naturally presented non-canonical circRNA-derived peptides

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: March 15, 2024

Circular RNAs (circRNAs) are covalently closed non-coding lacking the 5' cap and poly-A tail. Nevertheless, it has been demonstrated that certain circRNAs can undergo active translation. Therefore, aberrantly expressed in human cancers could be an unexplored source of tumor-specific antigens, potentially mediating anti-tumor T cell responses. This study presents immunopeptidomics workflow with a specific focus on generating circRNA-specific protein fasta reference. The main goal this is to streamline process identifying validating leukocyte antigen (HLA) bound peptides originating from circRNAs. We increase analytical stringency our by retaining identified independently two mass spectrometry search engines and/or applying group-specific FDR for canonical-derived circRNA-derived peptides. A subset specifically encoded region spanning back-splice junction (BSJ) validated targeted MS, direct Sanger sequencing respective transcripts. Our identifies 54 unique BSJ-spanning immunopeptidome melanoma lung cancer samples. approach enlarges catalog proteins explored immunotherapy.

Language: Английский

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Fragment ion intensity prediction improves the identification rate of non-tryptic peptides in timsTOF

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: May 10, 2024

Abstract Immunopeptidomics is crucial for immunotherapy and vaccine development. Because the generation of immunopeptides from their parent proteins does not adhere to clear-cut rules, rather than being able use known digestion patterns, every possible protein subsequence within human leukocyte antigen (HLA) class-specific length restrictions needs be considered during sequence database searching. This leads an inflation search space results in lower spectrum annotation rates. Peptide-spectrum match (PSM) rescoring a powerful enhancement standard searching that boosts performance. We analyze 302,105 unique synthesized non-tryptic peptides ProteomeTools project on timsTOF-Pro generate ground-truth dataset containing 93,227 MS/MS spectra 74,847 peptides, used fine-tune deep learning-based fragment ion intensity prediction model Prosit. demonstrate up 3-fold improvement identification immunopeptides, as well increased detection low input samples.

Language: Английский

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Analysis and Visualization of Quantitative Proteomics Data Using FragPipe-Analyst

Journal of Proteome Research, Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 10, 2024

The FragPipe computational proteomics platform is gaining widespread popularity among the research community because of its fast processing speed and user-friendly graphical interface. Although produces well-formatted output tables that are ready for analysis, there still a need an easy-to-use downstream statistical analysis visualization tool. FragPipe-Analyst addresses this by providing R shiny web server to assist users in conducting analyses resulting quantitative data. It supports major quantification workflows, including label-free quantification, tandem mass tags, data-independent acquisition. offers range useful functionalities, such as various missing value imputation options, data quality control, unsupervised clustering, differential expression (DE) using Limma, gene ontology pathway enrichment Enrichr. To support advanced customized visualizations, we also developed FragPipeAnalystR, package encompassing all functionalities extended site-specific post-translational modifications (PTMs). FragPipeAnalystR both open-source freely available.

Language: Английский

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diaTracer enables spectrum-centric analysis of diaPASEF proteomics data

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: Jan. 2, 2025

Abstract Data-independent acquisition has become a widely used strategy for peptide and protein quantification in liquid chromatography-tandem mass spectrometry-based proteomics studies. The integration of ion mobility separation into data-independent analysis, such as the diaPASEF technology available on Bruker’s timsTOF platform, further improves accuracy depth achievable using acquisition. We introduce diaTracer, spectrum-centric computational tool optimized data. diaTracer performs three-dimensional (mass to charge ratio, retention time, mobility) peak tracing feature detection generate precursor-resolved “pseudo-tandem spectra”, facilitating direct (“spectral-library free”) identification from is stand-alone fully integrated FragPipe platform. demonstrate performance data triple-negative breast cancer, cerebrospinal fluid, plasma samples, phosphoproteomics human leukocyte antigens immunopeptidomics experiments, low-input spatial study. also show that enables unrestricted post-translational modifications open/mass-offset searches.

Language: Английский

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Oktoberfest: Open‐source spectral library generation and rescoring pipeline based on Prosit

PROTEOMICS, Journal Year: 2023, Volume and Issue: 24(8)

Published: Sept. 6, 2023

Abstract Machine learning (ML) and deep (DL) models for peptide property prediction such as Prosit have enabled the creation of high quality in silico reference libraries. These libraries are used various applications, ranging from data‐independent acquisition (DIA) data analysis to data‐driven rescoring search engine results. Here, we present Oktoberfest, an open source Python package our spectral library generation pipeline originally only available online via ProteomicsDB. Oktoberfest is largely agnostic provides access predictions, promoting adoption state‐of‐the‐art ML/DL proteomics pipelines. We demonstrate its ability reproduce even improve results previously published analyses on two distinct use cases. freely GitHub ( https://github.com/wilhelm‐lab/oktoberfest ) can easily be installed locally through cross‐platform PyPI package.

Language: Английский

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De novo peptide sequencing with InstaNovo: Accurate, database-free peptide identification for large scale proteomics experiments

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Aug. 31, 2023

Abstract Bottom-up mass spectrometry-based proteomics is challenged by the task of identifying peptide that generates a tandem spectrum. Traditional methods rely on known sequence databases are limited and may not be applicable in certain contexts. De novo sequencing, which assigns sequences to spectra without prior information, valuable for various biological applications; yet, due lack accuracy, it remains challenging apply this approach many situations. Here, we introduce InstaNovo, transformer neural network with ability translate fragment ion peaks into amino acids make up studied peptide(s). The model was trained 28 million labelled matched 742k human peptides from ProteomeTools project. We demonstrate InstaNovo outperforms current state-of-the-art benchmark datasets showcase its utility several applications. Building upon intuition, also InstaNovo+, multinomial diffusion further improves performance iterative refinement predicted sequences. Using these models, could de antibody-based therapeutics unprecedented coverage, discover novel peptides, detect unreported organisms different datasets, thereby expanding scope detection rate searches. Finally, experimentally validate tryptic non-tryptic targeted proteomics, demonstrating fidelity our predictions. Our models unlock plethora opportunities across scientific domains, such as direct protein immunopeptidomics, exploration dark proteome.

Language: Английский

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MS²Rescore 3.0 Is a Modular, Flexible, and User-Friendly Platform to Boost Peptide Identifications, as Showcased with MS Amanda 3.0

Journal of Proteome Research, Journal Year: 2024, Volume and Issue: 23(8), P. 3200 - 3207

Published: March 16, 2024

Rescoring of peptide-spectrum matches (PSMs) has emerged as a standard procedure for the analysis tandem mass spectrometry data. This emphasizes need software maintenance and continuous improvement such algorithms. We introduce MS

Language: Английский

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What’s new in single-cell proteomics

Current Opinion in Biotechnology, Journal Year: 2024, Volume and Issue: 86, P. 103077 - 103077

Published: Feb. 14, 2024

Language: Английский

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