Cross-linking mass spectrometry analyses of three different kinetochore protein complexes (KMN, NDC80C, MIS12C) using an MS-cleavable cross-linker, BuUrBu (DSBU)

In order to develop a simplified cross-linking mass spectrometry protocol, we applied one-step size-exclusion chromatography (SEC) for peptide purification after proteolysis with Lys-C and trypsin. Three benchmark protein complexes (KMN, NDC80C, MIS12C) from human kinetochore were cross-linked with an MS-cleavable cross-linker (BuUrBu). After proteolysis, digested peptides were purified with SEC omitting the step of C18 column to avoid loss of cross-linked peptides. Our protocol showed improvement of existing protocols, and we could successfully identify over 700 cross-links per experiment of each of the three protein complexes. We also observed that about half of the non-redundant cross-links were not lysine-lysine cross-links but cross-links between lysine and side chains with alcohols (serine, threonine, tyrosine).