Developing an Acidic Residue Reactive and Sulfoxide-Containing MS-CleavableHomobifunctional Cross-Linker for Probing Protein–Protein Interactions

摘要

Cross-linking mass spectrometry (XL-MS) has become a powerful strategy for defining protein–protein interactions and elucidating architectures of large protein complexes. However, one of the inherent challenges in MS analysis of cross-linked peptides is their unambiguous identification. To facilitate this process, we have previously developed a series of amine-reactive sulfoxide-containing MS-cleavable cross-linkers. These MS-cleavable reagents have allowed us to establish a common robust XL-MS workflow that enables fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MSⁿ). Although amine-reactive reagents targeting lysine residues have been successful, it remains difficult to characterize protein interaction interfaces with little or no lysine residues. To expand the coverage of protein interaction regions, we present here the development of a new acidic residue-targeting sulfoxide-containing MS-cleavable homobifunctional cross-linker, dihydrazide sulfoxide (DHSO). We demonstrate that DHSO cross-linked peptides display thesame predictable and characteristic fragmentation pattern during collisioninduced dissociation as amine-reactive sulfoxide-containing MS-cleavablecross-linked peptides, thus permitting their simplified analysis andunambiguous identification by MSⁿ. Additionally,we show that DHSO can provide complementary data to amine-reactivereagents. Collectively, this work not only enlarges the range of theapplication of XL-MS approaches but also further demonstrates therobustness and applicability of sulfoxide-based MS-cleavability inconjunction with various cross-linking chemistries.