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The enrichable disuccinimidyl bissulfoxide (DSBSO) cross\u2010linker has enabled comprehensive XL\u2013MS studies of human cells. However, existing DSBSO workflows demand multi\u2010day sample preparation with high input requirements and provide insufficient detection sensitivity. Here, we systematically optimize the in solution and gas\u2010phase chemistry of azide\u2010A\u2010DSBSO\u2010based XL\u2013MS. Importantly, we reduce sample preparation time to 10 h and introduce StageTip\u2010based strong cation exchange (SCX) separation to concomitantly remove nonvolatile salts and interfering contaminants. Applying our streamlined SCX protocol to intact\n                    <jats:italic>Bacillus subtilis<\/jats:italic>\n                    reduced sample consumption 15\u2010fold compared to conventional size\u2010exclusion chromatography\u2010based azide\u2010A\u2010DSBSO XL\u2010MS and doubled the identification numbers, yielding 3,209 protein interactions at a 1% false\u2010discovery rate. These results illustrate that our optimized workflow unites speed, analytical depth, and resource efficiency, making XL\u2013MS amenable to high\u2010throughput interactome profiling of complex biological samples.\n                  <\/jats:p>","DOI":"10.1002\/anie.202518355","type":"journal-article","created":{"date-parts":[[2025,11,21]],"date-time":"2025-11-21T07:19:22Z","timestamp":1763709562000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Optimized In\u2010Solution and Gas\u2010Phase Chemistry Enables High\u2010Efficiency Interactome Mapping by DSBSO\u2010Based Cross\u2010Linking Mass Spectrometry"],"prefix":"10.1002","volume":"65","author":[{"given":"Pin\u2010Lian","family":"Jiang","sequence":"first","affiliation":[{"name":"Department of Structural Biology Leibniz\u2010Forschungsinstitut f\u00fcr Molekulare Pharmakologie (FMP) Berlin Germany"},{"name":"Institute of Chemistry and Biochemistry Freie 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