CHARACTERIZATION OF GLUTATHIONE CONJUGATES OF PYRROLYLATED AMINO-ACIDS AND PEPTIDES BY LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY AND TANDEM MASS-SPECTROMETRY WITH ELECTROSPRAY-IONIZATION

High-performance liquid chromatography (HPLC) coupled with electrospra y mass spectrometry (ES-MS) and tandem mass spectrometry (MS-MS) was u sed to identify the products formed upon reaction of lysine-containing peptides with the neurotoxicant 2,5-hexanedione (2,5-HD). In addition , secondary autoxidative reaction products of the resultant alkylpyrro les with the biological thiol, glutathione, were characterized. ES mas s spectra of the HPLC-separated conjugates showed intense [M+H](+) ion s as well as several ions formed by amide and C-S bond cleavage. The g lutathione conjugates of pyrrolylated amino acids and peptides were an alyzed by ES ionization and MS-MS, and product-ion spectra showed frag mentation pathways typical of glutathione conjugates. ES-MS-MS analysi s of a synthetic nonapeptide modeling a sequence found in neurofilamen t proteins showed pyrrole formation after incubation with 2,5-HD, and sequence ions were used to assign the position of the pyrrole adduct. Subsequent reaction of the pyrrolylated peptide with reduced glutathio ne was evidenced by a shift in m/z of the sequence ions of the reactio n products with or without prior methylation. The results demonstrate the utility of ES-MS and ES-MS-MS in the characterization of xenobioti c-modified peptides and confirm that stable pyrrole-thiol conjugates a re formed by the reaction of biological thiols with pyrrolylated pepti des.