All-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 settings liberates peptides for standard MS2-based peptide identification. We present an all-in-one method where retention time matches between the crosslink precursor and the liberated peptides establish linkage, and MS2 sequencing identifies the source-liberated peptides. We demonstrate that DC4, a very labile cleavable crosslinker, generates high-intensity peptides in-source. Crosslinks can be identified from these liberated peptides, as they are chromatographically well-resolved from monolinks. Using bovine serum albumin (BSA) as a crosslinking test case, we detect 27% more crosslinks with pseudo-MS3 over a best-in-class MS3 method. While performance is slightly lower for whole-cell lysates (generating two-thirds of the identifications of a standard method), we find that 60% of these hits are unique, highlighting the complementarity of the method.