Elevated hemoglobin adducts derived from crotonaldehyde in healthy smokers and oral cancer patients by nanoflow liquid chromatography tandem mass spectrometry☆

Abstract

Hemoglobin adducts derived from reactive chemicals have been used as exposure biomarkers in vivo. We previously identified and quantified adducted peptides derived from acrolein in human hemoglobin after trypsin digestion. In this study, we characterized the Schiff base and Michael adducts of crotonaldehyde in human hemoglobin after NaBH₄ reduction to the stable adducts with a respective mass increase of 54.0470 and 72.0575 Da, determined by high-resolution mass spectrometry. We developed a workflow based on nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry to simultaneously quantify 29 adducted peptides derived from acrolein and crotonaldehyde in one drop of blood from smoking oral cavity cancer patients, healthy smokers, and healthy nonsmokers. Levels of ten adducted peptides were significantly elevated in smokers, despite their cancer status, and the adduct levels correlate with the extent of cigarette smoking. Comparing the adduct levels at the same site, the Michael adduct of acrolein is much higher than that of crotonaldehyde. Multivariate analysis by orthogonal partial least squares discriminant analysis suggests that the Michel adducts of acrolein at α-Lys-7, α-His-50, β-Lys-17, and the Schiff base adduct of crotonaldehyde at β-Lys-59 are the predominate contributors. This is the first report on the structural characterization of human hemoglobin adducts of crotonaldehyde and the detection and quantification of these adducts in human subjects. Our results reveal that cigarette smoking plays a major role in forming these adducted peptides which might serve as potential biomarkers for exposure to acrolein and crotonaldehyde.