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 electrospray mass spectrometry (ES-MS) and tandem mass spectrometry (MS-MS) was used 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 alkylpyrroles with the biological thiol, glutathione, were characterized. ES mass spectra of the HPLC-separated conjugates showed intense [M+H]+ ions as well as several ions formed by amide and C-S bond cleavage. The glutathione conjugates of pyrrolylated amino acids and peptides were analyzed by ES ionization and MS-MS, and product-ion spectra showed fragmentation pathways typical of glutathione conjugates. ES-MS-MS analysis of a synthetic nonapeptide modeling a sequence found in neurofilament 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 glutathione was evidenced by a shift in m/z of the sequence ions of the reaction products with or without prior methylation. The results demonstrate the utility of ES-MS and ES-MS-MS in the characterization of xenobiotic-modified peptides and confirm that stable pyrrole-thiol conjugates are formed by the reaction of biological thiols with pyrrolylated peptides.
Zhu
M
M
Wadsworth Center, New York State Department of Health, Albany 12201-0509, USA.
DeCaprio
A P
AP
Hauer
C R
CR
Spink
D C
DC
D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry; D004563:Electrochemistry; D005978:Glutathione / Q000032:analysis* / Q000737:chemistry; D006588:Hexanones / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D009842:Oligopeptides / Q000032:analysis* / Q000737:chemistry; D010084:Oxidation-Reduction; D011758:Pyrroles / Q000032:analysis* / Q000737:chemistry
D000596:Amino Acids; D003432:Cross-Linking Reagents; D006588:Hexanones; D009842:Oligopeptides; D011758:Pyrroles; C011269:2,5-hexanedione; D005978:Glutathione; D008239:Lysine