Enrichment of cysteinyl adducts of human serum albumin

William E. Funk, He Li, Anthony T. Iavarone, Evan R. Williams, Jacques Riby, Stephen M. Rappaport*

*Corresponding author for this work

Research output: Contribution to journalArticle

34 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)61-68
Number of pages8
JournalAnalytical Biochemistry
Volume400
Issue number1
DOIs
StatePublished - May 1 2010

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Keywords

  • Adducts
  • Biomarkers
  • Enrichment
  • Human serum albumin
  • Mass spectrometry

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Funk, W. E., Li, H., Iavarone, A. T., Williams, E. R., Riby, J., & Rappaport, S. M. (2010). Enrichment of cysteinyl adducts of human serum albumin. Analytical Biochemistry, 400(1), 61-68. https://doi.org/10.1016/j.ab.2010.01.006