Determination of the Sites of 4-Hydroxy-2-nonenal Adduction to Protein by Electrospray Tandem Mass Spectrometry

Electrospray ionization mass spectrometry (ESI-MS) and ESI tandem MS methods for the determination of the sites of 4-hydroxy-2-nonenal (HNE) adduction to protein have been developed and exemplified by the characterization of HNE-adducted apomyoglobin. The procedure involves an initial analysis by ESI-MS of the adducted protein to determine the stoichiometry of HNE incorporation. The adducted protein is then subjected to proteolytic digestion, followed by direct analyses of the unfractionated digest by ESI-MS for the localization of the sites of HNE adduction. Proteolytic digestion using trypsin produces fragments of a suitable length for analysis by tandem MS with low-energy collision-induced dissociation. The components containing HNE-adducted histidine residues are identified by scanning for precursors of m/z 266, which corresponds to an immonium ion derived from HNE-adducted histidine. Last, product ion scanning of each modified tryptic fragment is performed to provide additional structural detail and ...