title: Enrichment of cysteinyl adducts of human serum albumin.
abstract: We report a method to enrich cysteinyl adducts of human serum albumin (HSA), representing biomarkers of exposure to systemic electrophiles. Because the major site of HSA adduction is the single free sulfhydryl group at Cys34, we used thiol-affinity resins to remove mercaptalbumin (i.e., unadducted HSA) from the cysteinyl adducts. Electrospray ionization mass spectrometry was used to detect mercaptalbumin and HSA-Cys34 modifications before and after enrichment of HSA. Differences in adduct content were detected across samples of freshly isolated, archived, and commercial HSA. Cysteinylated and glycosylated adducts were present in all samples, with abundances decreasing in the following order: commercial HSA>archived HSA>fresh HSA. After enrichment of HSA, mercaptalbumin was no longer observed in mass spectra. The ratios of HSA adducts post-/preenrichment, quantified via the Bradford assay and gel electrophoresis, were 0.029 mg adducts/mg HSA in fresh HSA and 0.323 mg adducts/mg HSA in archived HSA. The apparent elevation of adduct levels in archived samples could be due to differences in specimen preparation and storage rather than to differences in circulating HSA adducts. We conclude that thiol-affinity resins can efficiently remove mercaptalbumin from HSA samples prior to characterization and quantitation of protein adducts of reactive systemic electrophiles.
authors.affiliation: Gillings School of Global Public Health, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: William E
authors.initials: WE
authors.lastname: Funk
authors.forename: He
authors.initials: H
authors.lastname: Li
authors.forename: Anthony T
authors.initials: AT
authors.lastname: Iavarone
authors.forename: Evan R
authors.initials: ER
authors.lastname: Williams
authors.forename: Jacques
authors.initials: J
authors.lastname: Riby
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Rappaport
mesh_terms: D000328:Adult; D002846:Chromatography, Affinity; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D012709:Serum Albumin / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D012709:Serum Albumin; D013438:Sulfhydryl Compounds; C001702:mercaptoalbumin; D003545:Cysteine