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

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 to conventional size-exclusion chromatography-based azide-A-DSBSO XL-MS and doubled the identification numbers, yielding 3,209 protein interactions at a 1% false-discovery rate. These results illustrate that our optimized workflow unites speed, analytical depth, and resource efficiency, making XL-MS amenable to high-throughput interactome profiling of complex biological samples.