Rapid and In-Depth Coverage of the (Phospho-)Proteome With Deep Libraries and Optimal Window Design for dia-PASEF

Molecular & Cellular Proteomics, Год журнала: 2022, Номер 21(9), С. 100279 - 100279

Опубликована: Авг. 6, 2022

Язык: Английский

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

Nature Communications, Год журнала: 2023, Номер 14(1)

Опубликована: Июль 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

Язык: Английский

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An Introduction to Mass Spectrometry-Based Proteomics

Journal of Proteome Research, Год журнала: 2023, Номер 22(7), С. 2151 - 2171

Опубликована: Июнь 1, 2023

Mass spectrometry is unmatched in its versatility for studying practically any aspect of the proteome. Because foundations mass spectrometry-based proteomics are complex and span multiple scientific fields, can be perceived as having a high barrier to entry. This tutorial intended an accessible illustrated guide technical details relatively simple quantitative proteomic experiment. An attempt made explain relevant concepts those with limited knowledge basic understanding proteins. experimental overview provided, from beginning sample preparation analysis protein group quantities, explanations how data acquired, processed, analyzed. A selection advanced topics briefly surveyed works further reading cited. To conclude, brief discussion future given, considering next-generation sequencing technologies that may complement create fruitful proteomics.

Язык: Английский

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MSBooster: improving peptide identification rates using deep learning-based features

Nature Communications, Год журнала: 2023, Номер 14(1)

Опубликована: Июль 27, 2023

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, rescoring peptide-to-spectrum matches additional features incorporating deep learning-based predictions peptide properties, LC retention time, ion mobility, and spectra. We demonstrate the utility tandem with MSFragger Percolator, several different workflows, including nonspecific searches (immunopeptidomics), direct from data independent acquisition data, single-cell proteomics, generated an mobility separation-enabled timsTOF MS platform. MSBooster is fast, robust, fully integrated into widely used FragPipe

Язык: Английский

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The GAPDH redox switch safeguards reductive capacity and enables survival of stressed tumour cells

Nature Metabolism, Год журнала: 2023, Номер 5(4), С. 660 - 676

Опубликована: Апрель 6, 2023

Abstract Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is known to contain an active-site cysteine residue undergoing oxidation in response hydrogen peroxide, leading rapid inactivation of the enzyme. Here we show that human and mouse cells expressing a GAPDH mutant lacking this redox switch retain catalytic activity but are unable stimulate oxidative pentose phosphate pathway enhance their reductive capacity. Specifically, find anchorage-independent growth spheroids limited by elevation endogenous peroxide levels largely dependent on functional switch. Likewise, tumour vivo stress suppressed when disabled cells. The induction additional intratumoural chemo- or radiotherapy synergized with deactivation Mice exhibit altered fatty acid metabolism kidney heart, apparently compensation for lack Together, our findings demonstrate physiological pathophysiological relevance mammals.

Язык: Английский

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Robust dimethyl‐based multiplex‐DIA doubles single‐cell proteome depth via a reference channel

Molecular Systems Biology, Год журнала: 2023, Номер 19(9)

Опубликована: Авг. 21, 2023

Single-cell proteomics aims to characterize biological function and heterogeneity at the level of proteins in an unbiased manner. It is currently limited proteomic depth, throughput, robustness, which we address here by a streamlined multiplexed workflow using data-independent acquisition (mDIA). We demonstrate automated complete dimethyl labeling bulk or single-cell samples, without losing depth. Lys-N digestion enables five-plex quantification MS1 MS2 level. Because channels are quantitatively isolated from each other, mDIA accommodates reference channel that does not interfere with target channels. Our algorithm RefQuant takes advantage this confidently quantifies twice as many per single cell compared our previous work (Brunner et al, PMID 35226415), while allows routine analysis 80 cells day. Finally, combined spatial increase throughput Deep Visual Proteomics seven-fold for microdissection four-fold MS analysis. Applying primary cutaneous melanoma, discovered signatures within distinct tumor microenvironments, showcasing its potential precision oncology.

Язык: Английский

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Elucidating the cellular determinants of targeted membrane protein degradation by lysosome-targeting chimeras

Science, Год журнала: 2023, Номер 382(6668)

Опубликована: Окт. 19, 2023

Targeted protein degradation can provide advantages over inhibition approaches in the development of therapeutic strategies. Lysosome-targeting chimeras (LYTACs) harness receptors, such as cation-independent mannose 6–phosphate receptor (CI-M6PR), to direct extracellular proteins lysosomes. In this work, we used a genome-wide CRISPR knockout approach identify modulators LYTAC-mediated membrane human cells. We found that disrupting retromer genes improved target by reducing LYTAC recycling plasma membrane. Neddylated cullin-3 facilitated LYTAC-complex lysosomal maturation and was predictive marker for efficacy. A substantial fraction cell surface CI-M6PR remains occupied endogenous M6P-modified glycoproteins. Thus, M6P biosynthesis increased internalization LYTAC-target complexes. Our findings inform design strategies next-generation LYTACs elucidate aspects occupancy trafficking.

Язык: Английский

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Exploration of cell state heterogeneity using single-cell proteomics through sensitivity-tailored data-independent acquisition

Nature Communications, Год журнала: 2023, Номер 14(1)

Опубликована: Сен. 22, 2023

Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. The limited amount material encapsulated in a single cell however, raises significant technical challenges molecular profiling. Due extensive optimization efforts, single-cell proteomics by Mass Spectrometry (scp-MS) has emerged as powerful tool facilitate proteome profiling from ultra-low amounts input, although further development needed realize its full potential. To this end, we carry out comprehensive orbitrap-based data-independent acquisition (DIA) for proteomics. Notably, find difference between optimal DIA methods high- low-load samples. We improve our low-input method relying on high-resolution MS1 quantification, thus enhancing sensitivity more efficiently utilizing available mass analyzer time. With input tailored method, are able accommodate long injection times high resolution, while keeping the scan cycle time low enough ensure robust quantification. Finally, demonstrate capability approach mouse embryonic stem culture conditions, showcasing global proteomes highlighting differences key metabolic enzyme expression subclusters.

Язык: Английский

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The impact of immunopeptidomics: From basic research to clinical implementation

Seminars in Immunology, Год журнала: 2023, Номер 66, С. 101727 - 101727

Опубликована: Фев. 9, 2023

The immunopeptidome is the set of peptides presented by major histocompatibility complex (MHC) molecules, in humans also known as human leukocyte antigen (HLA), on surface cells that mediate T-cell immunosurveillance. a sampling cellular proteome and hence it contains information about health state cells. peptide repertoire influenced intra- extra-cellular perturbations - such case drug exposure, infection, or oncogenic transformation. Immunopeptidomics bioanalytical method which are extracted from biological samples analyzed high-performance liquid chromatography coupled to tandem mass spectrometry (MS), resulting deep qualitative quantitative snapshot immunopeptidome. In this review, we discuss published immunopeptidomics studies recent years, grouped into three main domains: i) basic, ii) pre-clinical iii) clinical research applications. We review selected fundamental processing presentation machinery, HLA restriction advanced our understanding various diseases, how exploration antigenic landscape allowed immune targeting at stage, paving way pioneering exploratory trials where directly implemented conception innovative treatments for cancer patients.

Язык: Английский

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

Molecular & Cellular Proteomics, Год журнала: 2024, Номер 23(2), С. 100712 - 100712

Опубликована: Янв. 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

Язык: Английский

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