Benchmarking of quantitative proteomics workflows for limited proteolysis mass spectrometry

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Item Type:	Article
Title:	Benchmarking of quantitative proteomics workflows for limited proteolysis mass spectrometry
Creators Name:	Koudelka, T., Bassot, C. and Piazza, I.
Abstract:	Limited proteolysis coupled with mass spectrometry (LiP-MS) has emerged as a powerful technique for detecting protein structural changes and drug-protein interactions on a proteome-wide scale. However, there is no consensus on the best quantitative proteomics workflow for analyzing LiP-MS data. In this study, we comprehensively benchmarked two major quantification approaches-data-independent acquisition (DIA) and tandem mass tag (TMT) isobaric labeling-in combination with LiP-MS, using a drug-target deconvolution assay as a model system. Our results show that while TMT labeling enabled the quantification of more peptides and proteins with lower coefficients of variation (CVs), DIA-MS exhibited greater accuracy in identifying true drug targets and stronger dose-response correlation in protein targets peptides. Additionally, we evaluated the performance of freely available (FragPipe) versus commercial (Spectronaut) software tools for DIA-MS analysis, revealing that the choice between precision (FragPipe) and sensitivity (Spectronaut) largely depends on the specific experimental context. Our findings underscore the importance of selecting the appropriate LiP-MS quantification strategy based on the study objectives. This work provides valuable guidelines for researchers in structural proteomics and drug discovery, and highlights how advancements in mass spectrometry instrumentation, such as the Astral mass spectrometer, may further improve sensitivity and protein sequence coverage, potentially reducing the need for TMT labeling.
Keywords:	LiP-MS, DIA-MS, TMT, FAIMS, Structural Proteomics, DIA Software Benchmarking, FragPipe
Source:	Molecular & Cellular Proteomics
ISSN:	1535-9484
Publisher:	Elsevier / American Society for Biochemistry and Molecular Biology
Page Range:	100945
Date:	13 March 2025
Official Publication:	https://doi.org/10.1016/j.mcpro.2025.100945
PubMed:	View item in PubMed

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