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.