Protein Interaction Crosslinking for Mass Spectrometry

Comparison of BSA crosslinking efficiency by SDS-PAGE. Different crosslinkers were incubated with BSA at molar excess of crosslinker to protein (e.g., 20- 100-, or 500-fold). Crosslinking efficiency is shown by decreased mobility by SDS-PAGE and varied by crosslinker type, solubility, and concentration.

BSA crosslinked peptide spectra identified by MS2-MS3 method and XLinkX using DSSO crosslinker. XlinkX software uses unique fragment ion patterns of MS-cleavable crosslinkers (purple annotation) to detect and filter crosslinked peptides for a crosslinked database search.

Graph showing number of BSA crosslinked peptides identified using different noncleavable (BS3, DSS) and cleavable (DSSO) crosslinkers for various MSn methods. Both BS3 and DSS had similar numbers of crosslinked peptides identified for collision-induced fragmentation (CID) and high-energy CID (HCD) methods, but DSSO identified fewer crosslinked peptides. Because DSSO is MS-cleavable, the MS2-MS3 method can be used to identify more crosslinks when a linear-peptide database search is used. All crosslinkers showed similar numbers of identified crosslinked peptides by electron-transfer and higher-energy collision dissociation (EThcD).