Cleavable Cross-Linkers Redefined by a Novel MS-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 MS-based approach for identifying cross-linked peptides. However, MS (MS/MS)-based approaches currently outperform approaches utilizing MS. We show here that the sensitivity and specificity of triggering MS have been hampered algorithmically. Our four-step MS-trigger algorithm greatly outperformed currently employed methods and comes close to reaching the theoretical limit.