Cleavable Cross-Linkers Redefined by a Novel MS 3 -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 3 -based approach for identifying cross-linked peptides. However, MS 2 (MS/MS)-based approaches currently outperform approaches utilizing MS 3 . We show here that the sensitivity and specificity of triggering MS 3 have been hampered algorithmically. Our four-step MS 3 -trigger algorithm greatly outperformed currently employed methods and comes close to reaching the theoretical limit.