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 to confirm the results obtained by precursor ion scanning. Application of this approach to the characterization of HNE-modified apomyoglobin indicated that there were between three and ten HNE adducts per protein molecule and that the adduction occurred solely to histidine residues.