Abstract
Cross-linking mass spectrometry is rapidly becoming a choice method for determining a protein's higher-order structure as well as capturing inter-protein interactions. In particular, diazirene-based photo-activatable cross-linkers, such as sulfo-SDA have been shown to be effective at generating high-density cross-linking data. Previously, we have shown that this method may be used to study binding orientation between two non-covalently linked complexes; however, several unexpected ions were noted in the MS2 spectra. In this study, the tandem mass spectrometry fragmentation patterns of sulfo-SDA-initiated cross-linked peptides under higher-energy collision induced (HCD), collision induced (CID) and electron transfer with supplementary HCD (EThcD) dissociations are discussed. The analysis revealed substantial insights into localising cross-linking sites, which is essential for accurate determination of protein higher-order structural characteristics.