Covalent Labeling with an α,β-Unsaturated Carbonyl Scaffold for Studying Protein Structure and Interactions by Mass Spectrometry

A new covalent labeling (CL) reagent based on an alpha,beta-unsaturated carbonyl scaffold has been developed for studying protein structure and protein-protein interactions when coupled with mass spectrometry. We show that this new reagent scaffold can react with up to 13 different types of residues on protein surfaces, thereby providing excellent structural resolution. To illustrate the value of this reagent scaffold, it is used to identify the residues involved in the protein-protein interface that is formed upon Zn(II) binding to the protein beta-2-microglobulin. The modular design of the alpha,beta-unsaturated carbonyl scaffold allows facile variation of the functional groups, enabling labeling kinetics and selectivity to be tuned. Moreover, by introducing isotopically enriched functional groups into the reagent structure, labeling sites can be more easily identified by MS and MS/MS. Overall, this reagent scaffold should be a valuable CL reagent for protein higher order structure characterization by MS.