A high-efficiency Azide-A-DSBSO cross-linking-MS protocol for identifying thousands of in vivo protein interactions in single day

Enrichable and mass spectrometry (MS)-cleavable crosslinkers have gained popularity in crosslinking-MS (XL-MS) in the past decade. The azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide (DSBSO) crosslinker offers both these advantages, enabling deep XL-MS interactomics studies in intact cells. However, current DSBSO XL-MS workflows take several days, which limits the experimental throughput. Here, we developed an in-cell DSBSO XL-MS protocol that allows sample ready for MS analysis within 8 hours. By systematically optimizing digestion, click chemistry enrichment, LC parameters, and MS fragmentation, we increased the number of crosslink spectrum matches (CSMs) from single-shot experiments more than twofold compared to previous single-shot measurements. This allowed us to efficiently generate deep protein-protein interaction datasets from intact human mitochondria, Bacillus subtilis, and HEK293T cells.