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bioRxiv · 10.1101/2025.11.11.687786

Advancing DIA-based Limited Proteolysis Workflows: introducing DIA-LiPA

Van Leene, C.·Araftpoor, E.·Staes, A.·Argentini, A.·Bühler, M.·Gevaert, K.
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Abstract

Limited proteolysis coupled to mass spectrometry (LiP-MS) probes protein conformational dynamics, but interpretation of LiP-MS data is complicated by heterogenous proteolytic cleavage patterns and missing data. Recent advances in data-independent acquisition (DIA) and machine learning-based search engines promise improved sensitivity and reproducibility, yet their performance in LiP-MS workflows remains underexplored. We systematically evaluated selected library-free DIA workflows using a rapamycin-treated human cell lysate and a yeast heat shock dataset, benchmarking DIA-NN and Spectronaut for identification depth, reproducibility and false discovery rate control. Our results show that library-free approaches achieve high sensitivity, eliminating the experimental overhead and sample requirements associated with empirical libraries. Building on these advances, we introduce a DIA-based Limited Proteolysis data Analysis pipeline (DIA-LiPA), a data analysis workflow tailored for LiP-MS data that integrates semi-tryptic- and tryptic-level precursor data and accounts for missingness to enable structural interpretation. Validation across multiple datasets confirmed that DIA-LiPA reproduces known structural signatures and uncovers additional regulatory patterns, providing a robust framework for mechanistic insights into protein dynamics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/687786v2_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@19fc486org.highwire.dtl.DTLVardef@1f74b7org.highwire.dtl.DTLVardef@127db11org.highwire.dtl.DTLVardef@19f5b88_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Van Leene, C., Araftpoor, E., Staes, A., Argentini, A., Bühler, M., Gevaert, K.. 2025-11-12. Advancing DIA-based Limited Proteolysis Workflows: introducing DIA-LiPA. https://doi.org/10.1101/2025.11.11.687786

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