title: Discovery and characterization of a photo-oxidative histidine-histidine cross-link in IgG1 antibody utilizing ¹⁸O-labeling and mass spectrometry.
abstract: 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, (18)O 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, elimination of a water molecule leads to the final adduct with a net mass increase of 14 Da. Our findings are consistent with this mechanism. Successful discovery of cross-linked His-His again demonstrates the broad applicability and utility of our XChem-Finder approach in the discovery and elucidation of protein cross-linking, particularly without a priori knowledge of the chemical nature and site of cross-linking.
authors.affiliation: Analytical Research and Development, Amgen , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.forename: Zhongqi
authors.initials: Z
authors.lastname: Zhang
authors.forename: Janet
authors.initials: J
authors.lastname: Cheetham
authors.forename: Da
authors.initials: D
authors.lastname: Ren
authors.forename: Zhaohui Sunny
authors.initials: ZS
authors.lastname: Zhou
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D007553:Isotope Labeling; D010084:Oxidation-Reduction; D010103:Oxygen Isotopes / Q000737:chemistry*; D010449:Peptide Mapping; D010777:Photochemistry
chemical_list: D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D010103:Oxygen Isotopes; D006639:Histidine
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