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Abstract No.: ThP-046
Session: Biomarkers
Presentation date: Thu, Aug 31, 2006
Presentation time: 14:30 – 16:00

Mass Spectrometric Identification of Albumin as a Protein Target of 4-hydroxy-trans-2-nonenal in Human Serum: A Potential Biomarker of Carbonylation Damage?

Giancarlo Aldini1, Luca Gamberoni1, Marica Orioli1, Luca Regazzoni1, Giangiacomo Beretta1, Roberto Maffei Facino1, Marina Carini1

1 Istituto Chimico Farmaceutico Tossicologico, University of Milan, Milan, Italy

Correspondence address: Giancarlo Aldini, University of Milan, Istituto Chimico Farmaceutico Tossicologico, viale Abruzzi 42, Milan, 20131 Italy.

Keywords: Aldehydes; Biomarkers; MS/MS, Liquid Chromatography; Protein.

Novel aspect: Mass spectrometric identification of human serum albumin adducted with 4-hydroxy-trans-2-nonenal as a potential biomarker of carbonylation damage.


4-hydroxy-trans-2-nonenal (HNE) is the most abundant and toxic α,β-unsaturated aldehyde generated through the β-cleavage of hydroperoxides from ω-6 PUFAs, which plays a role in a variety of pathophysiological processes.1 To better understand the physiopathological role of HNE as well as to discover new compounds acting as carbonyl-quenchers, a specific biomarker of systemic HNE formation is strictly needed. Although immunological evidences indicate that human serum albumin (HSA) modified by HNE is a promising biomarker,2 no detailed studies on the reaction between HSA and HNE have been up to now reported, nor on the chemical characterization of the protein adduct, which represents the aim of the present investigation. By HPLC analysis it was firstly found that HNE is rapidly quenched by HSA due to the covalent adduction to the different accessible nucleophilic residues of the protein, as demonstrated by 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). A novel LC-ESI-MS/MS approach was then applied to enzymatically digested HNE-modified HSA in order to characterize the covalent modifications, based on the following steps: 1) identification, in the HNE-treated samples, of peptides undergoing a significant consumption (10% set as threshold level); 2) prediction of the MW and the multicharged ions of the potential HNE Michael (MA) and/or Schiff base (SB) adducts on the basis of the sequence of quenched peptides; 3) reconstitution of the SIC traces using as filter ions the predicted ion values and identification of HNE-adducts when the SIC traces reconstituted for the predicted HNE-adducts show a well detectable peak in the HNE treated sample only; 4) peptide identification and characterization of the modification site by MS2 analysis. Eleven different HNE-adducts (8 MA and 3 SB) were identified, 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 sites were determined using the lower HNE concentration: Cys34 and His146 (HNE-Michael adducts), and Lys199 (Schiff base). In the second part of the work, human serum was incubated for 2hrs with 10 and 50 ÁM HNE (a concentration range found in plasma under oxidative stress conditions). Adducted peptides were determined by LC-ESI-MS/MS in TRM mode. The results, indicating a dose-dependent formation of Lys199, His146 and Cys34 adducted peptides, evidence that albumin can act as primary target of covalent adduction of HNE in the biological matrix. In conclusion, Lys199, His146 and Cys34 HNE-adducted peptides are suitable tags of HNE-adducted albumin and could be useful biomarkers of oxidative and carbonylation damage in humans.

1. G. Poli, et al., IUBMB Life 50, 315 (2000).
2. S. Toyokuni, et al., Antioxid. Redox Signal. 2, 681 (2000).