biostudies-literatureBrodie NINatural Sciences and Engineering Research Council of CanadaNCI NIH HHSCanarie2146-2155https://www.ebi.ac.uk/biostudies/studies/S-EPMC11250984biostudies-literatureUnknown34(10)Crosslinking mass spectrometry (XL-MS) supports structure analysis of individual proteins and highly complex whole-cell interactomes. The identification of crosslinked peptides from enzymatic digests remains challenging, especially at the cell level. Empirical methods that use gas-phase cleavable crosslinkers can simplify the identification process by enabling an MS³-based strategy that turns crosslink identification into a simpler problem of detecting two separable peptides. However, the method is limited to select instrument platforms and is challenged by duty cycle constraints. Here, we revisit a pseudo-MS³ concept that incorporates in-source fragmentation, where a fast switch between gentle high-transmission source conditions and harsher in-source fragmentation seJournal of the American Society for Mass SpectrometryAll-in-One Pseudo-MS³ Method for the Analysis of Gas-Phase Cleavable Protein Crosslinking Reactions.PMC11250984P01 CA092584RGPIN-2017-04879RS-326Crowder DABrodie NISarpe VSchriemer DfalseAll-in-One Pseudo-MS³ Method for the Analysis of Gas-Phase Cleavable Protein Crosslinking Reactions.Crosslinking mass spectrometry (XL-MS) supports structure analysis of individual proteins and highly complex whole-cell interactomes. The identification of crosslinked peptides from enzymatic digests remains challenging, especially at the cell level. Empirical methods that use gas-phase cleavable crosslinkers can simplify the identification process by enabling an MS³-based strategy that turns crosslink identification into a simpler problem of detecting two separable peptides. However, the method is limited to select instrument platforms and is challenged by duty cycle constraints. Here, we revisit a pseudo-MS³ concept that incorporates in-source fragmentation, where a fast switch between gentle high-transmission source conditions and harsher in-source fragmentation se2023-01-01T00:00:00Z2023 Oct2025-06-01T00:12:05.24Z2024-11-12T23:23:11.037ZS-EPMC112509843759016510.1021/jasms.3c00134