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Mass spectrometric characterization of covalent modification of human serum albumin by 4‐hydroxy‐ trans ‐2‐nonenal
Author(s) -
Aldini Giancarlo,
Gamberoni Luca,
Orioli Marica,
Beretta Giangiacomo,
Regazzoni Luca,
Maffei Facino Roberto,
Carini Marina
Publication year - 2006
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.1067
Subject(s) - chemistry , adduct , albumin , human serum albumin , electrospray ionization , covalent bond , serum albumin , oxidative stress , protein carbonylation , nucleophile , chromatography , mass spectrometry , biochemistry , lipid peroxidation , organic chemistry , catalysis
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.