1. Enabling Photoactivated Cross-Linking Mass Spectrometric Analysis of Protein Complexes by Novel MS-Cleavable Cross-Linkers
- Author
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Lan Huang, Craig B. Gutierrez, Clinton Yu, Scott D. Rychnovsky, Xiaorong Wang, Sadie F. DePeter, and Leah J. Salituro
- Subjects
MS-cleavable cross-linkers ,CID, collision-induced dissociation ,DSSO ,protein-protein interactions ,photocross-linking ,SDASO, Succinimidyl diazirine sulfoxide ,Biochemistry ,Mass Spectrometry ,LC-MSn, liquid chromatography multistage mass spectrometry ,Analytical Chemistry ,chemistry.chemical_compound ,BMSO, Bismaleimide sulfoxide, a.k.a. 3,3'-sulfinylbis(N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)propanamide) ,PIP, proteasome-interacting protein ,19S regulatory particle ,SDASO-M, Succinimidyl diazirine sulfoxide (medium) aka. 2,5-Dioxopyrrolidin-1-yl 3-((3-(3-methyl-3H-diazirin-3-yl)propyl)sulfinyl)propanoate ,26S proteasome ,XL-MS ,SDASO-L, Succinimidyl diazirine sulfoxide (long) aka. 2,5-Dioxopyrrolidin-1-yl 3-((2-(3-(3-methyl-3H-diazirin-3-yl)propanamido)ethyl)sulfinyl) propanoate ,Chromatography ,Liquid ,0303 health sciences ,Chemistry ,030302 biochemistry & molecular biology ,Technological Innovation and Resources ,Serum Albumin, Bovine ,Bovine ,Photochemical Processes ,Mass spectrometric ,XL-MS, cross-linking mass spectrometry ,Cross-Linking Reagents ,Diazirine ,Biochemistry & Molecular Biology ,Proteasome Endopeptidase Complex ,PPI, Protein-protein interaction ,Collision-induced dissociation ,Saccharomyces cerevisiae ,Mass spectrometry ,Protein–protein interaction ,Fungal Proteins ,03 medical and health sciences ,NHS-diazirine ,19S RP, 19S regulatory particle ,SDASO ,Molecular Biology ,Serum Albumin ,20S CP, 20S core particle ,030304 developmental biology ,protein complexes ,DHSO, Dihydrazide sulfoxide, a.k.a., 3,3’-sulfinyldi(propanehydrazide) ,SDASO-S, Succinimidyl diazirine sulfoxide (short) aka. 2,5-Dioxopyrrolidin-1-yl 3-((2-(3-methyl-3H-diazirin-3-yl)ethyl)sulfinyl)propanoate ,Combinatorial chemistry ,Diazomethane ,MS, mass spectrometry ,Proteasome ,diazirine labeling ,BSA, bovine serum albumin ,Generic health relevance ,DSSO, Disuccinimidyl sulfoxide ,Multistage mass spectrometry ,Chromatography, Liquid ,MSn, multistage mass spectrometry - 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., Graphical Abstract, Highlights • Development of three mass spectrometry-cleavable photoreactive cross-linkers. • Characterization of diazirine-based photocross-linking with a standard protein BSA. • MSn-based workflow for photocross-linking analysis of protein complexes. • SDASO cross-linking mass spectrometry analyses of the yeast 26S proteasome complex., In Brief Although photochemistry complements residue-specific chemistry through labeling amino acids nonspecifically, existing photo-cross-linking reagents are thus far inapplicable to multisubunit protein complexes owing to low yields and high complexities of photo-cross-linked products. The development of the three sulfoxide-containing MS-cleavable photoreactive SDASO cross-linkers permits MSn-based analytical workflow for accurate identification of photo-cross-linked peptides, enabling complex PPI profiling for the first time. This work has established a solid foundation for future applications of photo-cross-linking in complex XL-MS studies.
- Published
- 2021