Optimized In-Solution and Gas-Phase Chemistry Enables High-Efficiency Interactome Mapping by DSBSO-Based
Pin-Lian Jiang1,2, Ying Zhu1,3, Jiaxin Cai1,3
1Department of Structural Biology, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany.
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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.
