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 m ethods for the determination of the sites of 4-hydroxy-2-nonenal (HNE) adduction to protein have been developed and exemplified by the chara cterization of HNE-adducted apomyoglobin, The procedure involves an in itial analysis by ESI-MS of the adducted protein to determine the stoi chiometry of HNE incorporation. The adducted protein is then subjected to proteolytic digestion, followed by direct analyses of the unfracti onated digest by ESI-MS for the localization of the sites of HNE adduc tion, Proteolytic digestion using trypsin produces fragments of a suit able length for analysis by tandem MS with low-energy collision-induce d dissociation. The components containing HNE-adducted histidine resid ues are identified by scanning for precursors of m/z 266, which corres ponds to an immonium ion derived from HNE-adducted histidine. Last, pr oduct ion scanning of each modified tryptic fragment is performed to p rovide additional structural detail and to confirm the results obtaine d by precursor ion scanning. Application of this approach to the chara cterization of HNE-modified apomyoglobin indicated that there were bet ween three and ten HNE adducts per protein molecule and that the adduc tion occurred solely to histidine residues.