Cysteine-enabled cleavability to advance cross-linking mass spectrometry for global analysis of endogenous protein-protein interactions

Cross-linking mass spectrometry (XL-MS) is a powerful technology for probing protein-protein interactions (PPIs) and elucidating architectures of protein complexes at the systems level. While successful, the proteome coverage remains limited. To expand the scope of global PPI profiling, we introduce an innovative cysteine-based cleavable XL-MS platform using non-cleavable heterobifunctional lysine-cysteine (K-C) cross-linkers. The oxidation-induced transformation of cysteine cleavability enables unambiguous identification of cross-linked peptides. This strategy has been successfully applied to proteome-wide XL-MS analysis of intact cells, and its broad applicability has been demonstrated using three heterobifunctional cross-linkers. A total of 25,401 unique linkages from 2007 proteins have been identified, significantly expanding the existing XL-PPI map and increasing the interconnectivity of the human interactome. The XL-data generated here has been coupled with AlphaFold-based predictions and integrative modeling to reveal the structural characteristics of cellular networks, offering insights into the organization of native protein complexes in cells including the SERBP1-ribosome and eEF1A1-eEF1B complexes. Due to the effectiveness in modulating cysteine cleavability, our work presents an avenue for developing bifunctional/multifunctional cross-linking reagents to further advance XL-MS technologies. Additionally, the same strategy can be easily adapted to facilitate the characterization of cysteine modifications, reactivity and interactions to benefit chemical proteomics.