Cross-linking mass spectrometry is an emergent technology for interactomics and structural biology1. Chemical cross-linking enables the capture of protein-protein interactions (PPIs) in native cellular environments, and the identification of cross-linked peptides permits the determination of both identity and connectivity of PPIs. In addition, the resulting cross-links can be utilized as distance constraints for various applications, ranging from structure validation and integrative modeling to de novo structure prediction. The development of sulfoxide-containing MS-cleavable cross-linkers enables fast, simplified and accurate identification of cross-linked peptides to significantly advance targeted and proteome-wide XL-MS studies for PPI mapping in vitro and in vivo.
These homobifunctional sulfoxide-containing MS-cleavable cross-linkers possess two symmetric C-S bonds that can be preferentially cleaved in the gas phase using collision-induced dissociation (CID) during tandem mass spectrometry (MS/MS or MS2). This results in the physical separation of a cross-link to yield unique peptide fragment pairs with a defined mass relationship1-7. These characteristic and predictable MS2 fragment ion pairs are then subjected to MS3 analysis for simplified and unambiguous identification of cross-linked peptides by conventional database searching tools. The MSn-based workflow provides fast and accurate cross-link identification at various scales including systems-wide analysis. In addition, these MS-cleavable cross-linkers provide the flexibility of allowing MS2-based and MS2-MS3-combined workflows for cross-link identification by implementing higher-energy collisional dissociation (HCD) and/or electron transfer dissociation (ETD)1.
The isotope-coded cross-linkers (d0/d10-DMDSSO) can be used for pair-wise comparison of conformational changes of protein complexes1, 8. All of the listed cross-linkers can be coupled with isobaric labeling reagents (e.g. TMT) for multiplexed quantitative XL-MS analyses1,9. In addition, parallel reaction monitoring (PRM) can be employed for targeted quantitative XL-MS analyses10.
Cross-linkers with distinct cross-linking chemistries (e.g. DSSO, DHSO, and BMSO) can be integrated for combinatory XL-MS analyses to expand PPI coverage and facilitate structure modeling of protein complexes10.
These reagents have been successfully applied to map PPIs at the proteome-wide scale and elucidate architectures of protein complexes in vivo and in vitro.