Optimized In-Solution and Gas-Phase Chemistry Enables High-Efficiency Interactome Mapping by DSBSO-Based Cross-Linking Mass Spectrometry.

ABSTRACT: Cross-linking mass spectrometry (XL-MS) allows characterizing protein structures and interactions in highly complex samples. The enrichable disuccinimidyl bissulfoxide (DSBSO) cross-linker has enabled comprehensive XL-MS studies of human cells. However, existing DSBSO workflows demand multi-day sample preparation with high input requirements and provide insufficient detection sensitivity. Here, we systematically optimize the in solution and gas-phase chemistry of azide-A-DSBSO-based XL-MS. Importantly, we reduce sample preparation time to 10 h and introduce StageTip-based strong cation exchange (SCX) separation to concomitantly remove nonvolatile salts and interfering contaminants. Applying our streamlined SCX protocol to intact Bacillus subtilis reduced sample consumption 15-fold compared

SUBMITTER: Jiang PL 

PROVIDER: S-EPMC12828450 | biostudies-literature | 2026 Jan

REPOSITORIES: biostudies-literature