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.