Abstract
Cross-linking mass spectrometry (XL-MS) is a powerful tool for probing protein structures and protein-protein interactions. While chemical cross-linkers target specific residues with defined chemistry, photo-cross-linkers offer superior reactivity but have been hampered by incomplete mechanistic understanding and lack of robust analytical framework. Here, we demonstrate that diazirine-based photo-cross-links are inherently MS-cleavable, generating composite backbone and side-chain fragments, which have nevertheless confounded spectral interpretation. Yet by leveraging the side-chain fragmentation fingerprints (sFFP), we develop a machine learning model and subsequently, a rule-based filtering algorithm. When integrated with existing search platforms, our workflow significantly improves ion coverage and reduces false discovery rate for site identification. We further develop a homo-bifunctional diazirine cross-linker, allowing for cross-linking on-demand. This reagent captures transient tetrameric assemblies of human HSP90β and reveals structural transitions in association equilibrium under heat stress, details otherwise inaccessible with chemical cross-linking. Together, this work establishes a transformative framework in XL-MS, combining the temporal resolution of photo-activation with analytical confidence for residue-level structural insights.
Acknowledgements
We are grateful to Prof. Si-min He and the pFind team for their technical guidance. We also thank Prof. Chengdong Huang for kindly providing purified HSP90β protein, and Drs. Dingfei Yan and Haiteng Deng from the MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, for their expert assistance with mass spectrometry analysis at the Center of Protein Analysis Technology. In addition, we acknowledge the support of the Proteomics Technology Platform at the National Center for Protein Sciences (Beijing) located in the Changping Phoenix Facility, as well as the State Key Laboratory of Natural and Biomimetic Drugs at Peking University, for providing mass spectrometry services, and Dr. Xiaohui Zhang for experimental assistance and spectroscopic support.
Funding
This work was supported by the National Natural Science Foundation of China (Grant 224B2401 to Y.J. and Grant 92353304 to C.T.) and the National Key Research and Development Program of China (Grant 2023YFF1204400 to C.T.).
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Jiang, Y., Zhao, R., Mao, P. et al. Inherent MS-cleavability of diazirine photo-cross-links enables residue-level structural analysis. Nat Commun (2026). https://doi.org/10.1038/s41467-026-73272-0
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DOI: https://doi.org/10.1038/s41467-026-73272-0
