Research Article

Mass spectrometric characterization of covalent modification of human serum albumin by 4-hydroxy-trans-2-nonenal

  1. Giancarlo Aldini*,
  2. Luca Gamberoni,
  3. Marica Orioli,
  4. Giangiacomo Beretta,
  5. Luca Regazzoni,
  6. Roberto Maffei Facino,
  7. Marina Carini

Article first published online: 3 AUG 2006

DOI: 10.1002/jms.1067

Issue

Journal of Mass Spectrometry

Journal of Mass Spectrometry

Volume 41, Issue 9, pages 1149–1161, September 2006

Additional Information

How to Cite

Aldini, G., Gamberoni, L., Orioli, M., Beretta, G., Regazzoni, L., Maffei Facino, R. and Carini, M. (2006), Mass spectrometric characterization of covalent modification of human serum albumin by 4-hydroxy-trans-2-nonenal. J. Mass Spectrom., 41: 1149–1161. doi: 10.1002/jms.1067

Author Information

  1. Istituto di Chimica Farmaceutica e Tossicologica ‘Pietro Pratesi’, Faculty of Pharmacy, University of Milan, Viale Abruzzi 42, I-20131 Milan, Italy

*Istituto di Chimica Farmaceutica e Tossicologica ‘Pietro Pratesi’, Faculty of Pharmacy, University of Milan Viale Abruzzi 42, 20131 Milan, Italy.

Publication History

  1. Issue published online: 11 SEP 2006
  2. Article first published online: 3 AUG 2006
  3. Manuscript Accepted: 22 MAY 2006
  4. Manuscript Received: 18 MAR 2006

Funded by

  • COFIN2004 (Cofinanziamento Programma Nazionale 2004)

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Keywords:

  • human serum albumin;
  • 4-hydroxy-trans-2-nonenal;
  • Michael and Schiff base adduction;
  • ESI-MS direct infusion;
  • LC-ESI-MS/MS analysis

Abstract

Several pieces of evidence indicate that albumin modified by HNE is a promising biomarker of systemic oxidative stress and that HNE-modified albumin may contribute to the immune reactions triggered by lipid peroxidation-derived antigens. In this study, we found by HPLC analysis that HNE is rapidly quenched by human serum albumin (HSA) because of the covalent adduction to the different accessible nucleophilic residues of the protein, as demonstrated by electrospray ionization mass spectrometry (ESI-MS) direct infusion experiments (one to nine HNE adducts, depending on the molar ratio used, from 1 : 0.25 to 1 : 5 HSA : HNE). An LC-ESI-MS/MS approach was then applied to enzymatically digested HNE-modified albumin, which permitted the identification of 11 different HNE adducts, 8 Michael adducts (MA) and 3 Schiff bases (SB), involving nine nucleophilic sites, namely: His67 (MA), His146 (MA), His242 (MA), His288 (MA), His510 (MA), Lys 195 (SB), Lys 199 (MA, SB), Lys525 (MA, SB) and Cys34 (MA). The most reactive HNE-adduction site was found to be Cys34 (MA) followed by Lys199, which primarily reacts through the formation of a Schiff base, and His146, giving the corresponding HNE Michael adduct. These albumin modifications are suitable tags of HNE-adducted albumin and could be useful biomarkers of oxidative and carbonylation damage in humans. Copyright © 2006 John Wiley & Sons, Ltd.

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