biostudies-literatureKolbowski LDeutsche ForschungsgemeinschaftWellcome Trust15461-15464https://www.ebi.ac.uk/biostudies/studies/S-EPMC10603603biostudies-literatureUnknown95(42)Cross-linking mass spectrometry (MS) is currently transitioning from a routine tool in structural biology to enabling structural systems biology. MS-cleavable cross-linkers could substantially reduce the associated search space expansion by allowing a MS³-based approach for identifying cross-linked peptides. However, MS² (MS/MS)-based approaches currently outperform approaches utilizing MS³. We show here that the sensitivity and specificity of triggering MS³ have been hampered algorithmically. Our four-step MS³-trigger algorithm greatly outperformed currently employed methods and comes close to reaching the theoretical limit.Analytical chemistryCleavable Cross-Linkers Redefined by a Novel MS³-Trigger Algorithm.PMC10603603EXC 2008390540038449713269203149Rappsilber JFischer LKolbowski LfalseCleavable Cross-Linkers Redefined by a Novel MS³-Trigger Algorithm.Cross-linking mass spectrometry (MS) is currently transitioning from a routine tool in structural biology to enabling structural systems biology. MS-cleavable cross-linkers could substantially reduce the associated search space expansion by allowing a MS³-based approach for identifying cross-linked peptides. However, MS² (MS/MS)-based approaches currently outperform approaches utilizing MS³. We show here that the sensitivity and specificity of triggering MS³ have been hampered algorithmically. Our four-step MS³-trigger algorithm greatly outperformed currently employed methods and comes close to reaching the theoretical limit.2023-01-01T00:00:00Z2023 Oct2026-05-28T08:32:06.964Z2025-02-18T23:29:47.425ZS-EPMC106036033781615510.1021/acs.analchem.3c01673