Discovery and Characterization of a Photo-OxidativeHistidine-Histidine Cross-Link in IgG1 Antibody Utilizing 18O-Labeling and Mass Spectrometry

摘要

A novel photo-oxidative cross-linking between two histidines (His-His) has been discovered and characterized in an IgG1 antibody via the workflow of XChem-Finder, ¹⁸O labeling and mass spectrometry (Anal. Chem. 2013, 85, 5900−5908 [] [] []). Its structure was elucidated by peptide mapping with multiple proteases with various specificities (e.g., trypsin, Asp-N, and GluC combined with trypsin or Asp-N) and mass spectrometry with complementary fragmentation modes (e.g., collision-induced dissociation (CID) and electron-transfer dissociation (ETD)). Our data indicated that cross-linking occurred across two identical conserved histidine residues on two separate heavy chains in the hinge region, which is highly flexible and solvent accessible. On the basis of model studies with short peptides, it has been proposed that singlet oxygen reacts with the histidyl imidazole ring to form an endoperoxide and then converted to the 2-oxo-histidine (2-oxo-His) and His+32 intermediates, the latter is subject to a nucleophilic attack by the unmodified histidine; and finally, eliminationof a water molecule leads to the final adduct with a net mass increaseof 14 Da. Our findings are consistent with this mechanism. Successfuldiscovery of cross-linked His-His again demonstrates the broad applicabilityand utility of our XChem-Finder approach in the discovery and elucidationof protein cross-linking, particularly without a priori knowledge of the chemical nature and site of cross-linking.