Improved Cross-Linking Coverage for Protein Complexes Containing Low Levels of Lysine by Using an Enrichable Photo-Cross-Linker

Chemical cross-linking coupled with mass spectrometry(XL-MS) isan important technique for the structural analysis of protein complexeswhere the coverage of amino acids and the identification of cross-linkedsites are crucial. Photo-cross-linking has multisite reactivity andis valuable for the structural analysis of chemical cross-linking.However, a high degree of heterogeneity results from this multisitereactivity, which results in samples with higher complexity and lowerabundance. Additionally, the applicability of photo-cross-linkingis limited to purified protein complexes. In this work, we demonstratea photo-cross-linker, alkynyl-succinimidyl-diazirine (ASD) with thereactive groups of N-hydroxysuccinimide ester anddiazirine, as well as the click-enrichable alkyne group. Photo-cross-linkerscan provide higher site reactivity for proteins that contain onlya small number of lysine residues, thereby complementing the morecommonly used lysine-targeting cross-linkers. By systematically analyzingproteins with differing lysine contents and differing flexibilities,we demonstrated clear enhancement in structure elucidation for proteinscontaining less lysine and with high flexibility. In addition, enrichmentapproaches of alkynyl-azide click chemistry conjugated with biotin-streptavidinpurification (coinciding with parallel orthogonal digestion) improvedthe identification coverage of cross-links. We show that this photo-cross-linkingapproach can be used for membrane proteome-wide complex analysis.This method led to the identification of a total of 14066 lysine-Xcross-linked site pairs from a total of 2784 proteins. Thus, thiscross-linker is a valuable addition to a photo-cross-linking toolkitand improves the identification coverage of XL-MS in functional structureanalysis.

Automated summary

Chemical cross-linking coupled with mass spectrometry (XL-MS) is a crucial technique for the structural analysis of protein complexes. Photo-cross-linking, although heterogenous, is valuable for this analysis. In this study, a photo-cross-linking method using alkynyl-succinimidyl-diazirine (ASD) was demonstrated to enhance structure elucidation for proteins with less lysine and high flexibility. Additionally, enrichment approaches improved cross-link identification coverage. This method can be used for membrane proteome-wide complex analysis and significantly improves XL-MS in functional structure analysis.