1. Enabling Photoactivated Cross-Linking Mass Spectrometric Analysis of Protein Complexes by Novel MS-Cleavable Cross-Linkers.
- Author
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Gutierrez, Craig, Salituro, Leah J, Yu, Clinton, Wang, Xiaorong, DePeter, Sadie F, Rychnovsky, Scott D, and Huang, Lan
- Subjects
26S proteasome ,DSSO ,MS-cleavable cross-linkers ,NHS-diazirine ,SDASO ,XL-MS ,diazirine labeling ,photocross-linking ,protein complexes ,protein-protein interactions ,Biochemistry & Molecular Biology - Abstract
Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. While residue-specific XL-MS studies have been very successful, accessibility of interaction regions nontargetable by specific chemistries remain difficult. Photochemistry has shown great potential in capturing those regions because of nonspecific reactivity, but low yields and high complexities of photocross-linked products have hindered their identification, limiting current studies predominantly to single proteins. Here, we describe the development of three novel MS-cleavable heterobifunctional cross-linkers, namely SDASO (Succinimidyl diazirine sulfoxide), to enable fast and accurate identification of photocross-linked peptides by MSn. The MSn-based workflow allowed SDASO XL-MS analysis of the yeast 26S proteasome, demonstrating the feasibility of photocross-linking of large protein complexes for the first time. Comparative analyses have revealed that SDASO cross-linking is robust and captures interactions complementary to residue-specific reagents, providing the foundation for future applications of photocross-linking in complex XL-MS studies.
- Published
- 2021