Strategies for Acid and Amine Cross-linking and Labeling for Protein Structural Characterization Using Mass Spectrometry

Cross-linking and covalent labeling are common tools for protein structural elucidation by mass spectrometry. However, despite the importance of electrostatic interactions in proteins, there are not many tools for probing both acids and amines. Therefore, we introduce novel solution-phase amine-to-acid cross-linkers and single reagent amine and acid covalent labels utilizing carbodiimide chemistry. Cross-linking and labeling sites were determined using top-down and enzymatic digestion approaches. Ubiquitin was chosen as a model protein for isotopically labeled ¹³C–¹⁵N-glycine cross-linking/covalent labeling and glycyl-l-proline cross-linking/covalent labeling with top-down mass spectrometry as a proof of concept. Alpha synuclein, an intrinsically disordered protein that plays a role in multiple neurological disorders, was also cross-linked/covalent labeled with these methods/reagents. We expect that these structural characterization techniques will play a role in gaining insight into functionally and pathologically important structural tendencies of disordered proteins.