Dimethyl labeling of N-terminal amines allows unambiguous identification of protein crosslinks

Protein crosslinks induced through either deliberate enzymatic oxidation or reactive oxidants (oxidative eustress/distress), are associated with multiple human pathologies including atherosclerosis, Alzheimer's and Parkinson's diseases. In many cases, the nature of the crosslinks, their position(s) either within (intramolecular) or between (intermolecular) polypeptide chains, and concentrations are unclear. Although limited data are available from specific antibodies, detailed characterization of protein crosslinks is often performed by mass spectrometric analysis of peptides from proteolytic digestion. Such analyses are challenging due to the low concentration of these species, and the complexity of their fragment ion spectra when compared to non-crosslinked species. We hypothesized that highly efficient and specific chemical amine labeling of the two N-termini in crosslinked peptides (compared to the single N-terminus of linear peptides), using "light" and "heavy" isotope-labelled reagents would facilitate identification, validation and quantification of crosslinks. This method was compared to a previous enzyme-catalyzed ¹⁸O C-terminal carboxylate labeling approach. N-terminal amine dimethyl labeling is shown to have major advantages over the ¹⁸O-approach including high labeling yields (92-100 %) and well-defined mass spectrometric isotope distribution patterns. This approach has allowed identification of novel dityrosine crosslinks between pair of tyrosine (Tyr, Y) residues in photo-oxidized β-casein (Y195-Y195, Y195-Y208, Y208-Y208), and α-synuclein exposed to nitrosative stress (Y39-Y39, Y39-Y125, Y39-Y133, Y133-Y136). This approach is also applicable to disulfide bond mapping, with 15 of 17 disulfides in serum albumin readily detected. These data indicate that dimethyl labeling is a highly versatile and efficient approach for the site-specific identification of oxidation- and nitration-induced crosslinks in proteins.