Enabling Photoactivated Cross-Linking Mass Spectrometric Analysis of Protein Complexes by Novel MS-Cleavable Cross-Linkers

Craig Gutierrez,Leah J Salituro,Clinton Yu,Xiaorong Wang,Sadie F Depeter,Scott D Rychnovsky,Lan Huang

doi:10.1016/j.mcpro.2021.100084

Craig Gutierrez, Leah J Salituro + Show 5 more

Open Access

https://doi.org/10.1016/j.mcpro.2021.100084

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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.

Highlights

Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein–protein interactions and elucidating architectures of protein complexes
Our results demonstrate that MS-cleavability enables accurate identification of photocross-linked peptides and that the Succinimidyl diazirine sulfoxide (SDASO)-based cross-linking mass spectrometry (XL-MS) workflow is well-suited for probing Protein-protein interaction (PPI) in complex samples
We constructed three MS-cleavable heterobifunctional SDASO cross-linkers composed of a fixed NHS ester end and a flexible diazirine side with varying lengths from the center sulfoxide, well within the distance range suited for studying PPIs [2] (Fig. 1, B–D)

Summary

Graphical Abstract

Photochemistry complements residue-specific chemistry through labeling amino acids nonspecifically, existing photo-cross-linking reagents are far inapplicable to multisubunit protein complexes owing to low yields and high complexities of photo-cross-linked products. We describe the development of three novel MS-cleavable heterobifunctional crosslinkers, namely SDASO (Succinimidyl diazirine sulfoxide), to enable fast and accurate identification of photocrosslinked 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. Integration of multiple cross-linkers has improved characterization of PPIs and increased the depth and accuracy of structural analysis [7, 8, 18, 19], demonstrating the benefits of multichemistrybased combinatory XL-MS approaches Despite these successes, mapping interaction regions lacking targetable residues by specific chemistry remains challenging. Comparison with residue-specific XL-MS data has determined that SDASO cross-linking is robust and captures PPIs complementary to existing reagents

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RESULTS

DISCUSSION