Characterization and Quantification of Acrolein-Induced Modifications in Hemoglobin by Mass Spectrometry─Effect of Cigarette Smoking

Abstract

Exposure to acrolein, the smallest α, β-unsaturated aldehyde, in humans originates from cigarette smoking and other environmental sources, food cooking, and endogenous lipid peroxidation and metabolism. The protein modification caused by acrolein is associated with various diseases, including cancer, cardiovascular, and neurodegenerative diseases. In this study, acrolein-modified human hemoglobin was reduced by sodium borohydride. Thus, three types of modifications, that is, Schiff base, Michael addition, and formyl-dehydropiperidion adducts, were converted to the corresponding stable adducts, leading to mass increases of 40.0313, 58.0419, and 96.0575 Da, respectively. These stable acrolein-modified hemoglobin peptides were identified by nanoflow liquid chromatography coupled to a high-resolution nanoelectrospray ionization tandem mass spectrometry. Among the 26 different types and sites of modifications, 15 of them showed a dose-dependent increase with increasing concentrations of acrolein. To investigate the role of acrolein-induced modifications in smoking and oral cancer, the 15 dose–responsive acrolein-modified peptides, together with three ethylated peptides previously identified, were quantified in oral cancer patients, healthy smokers, and healthy nonsmokers. The results reveal that the relative extents of the Michael-type adduct at α-Lys-16, α-His-50, and β-Lys-59 are significantly higher in smokers (oral cancer and healthy) than in nonsmokers. Areas under the receiver operating characteristic curve of these peptides range from 0.7500 to 0.9375, indicating the ability to discriminate smokers from nonsmokers. Additionally, these acrolein-modified peptides correlate with three ethylated peptides at the N-termini of α- and β-globin, as well as β-His-77, and with the number of cigarettes smoked per day. Therefore, measuring the reduced Michael adducts at α-Lys-16, α-His-50, and β-Lys-59 of hemoglobin from one drop of blood by this sensitive and specific method may reflect the increase of acrolein exposure due to cigarette smoking.

The Tobacco Smoke Component, Acrolein, as a Major Culprit in Lung Diseases and Respiratory Cancers: Molecular Mechanisms of Acrolein Cytotoxic Activity

“…Over the past few decades, based on the analysis of the available literature, it can be undeniably stated that the basis of the harmful effects of acrolein in the pathology of many diseases, i.e., neurodegenerative diseases, heart diseases, cancer or lung diseases, is the aldehyde’s ability to extremely strongly interact with numerous proteins of our body that perform various regulatory, homeostatic or immunological functions [ 13 , 15 ]. The latest research by Chen et al [ 67 ] indicates that acrolein interacts with α-Lys-16, α-His-50, and β-Lys-59 of smokers’ hemoglobin by changing the protein structure. The increase in the levels of acrolein-modified peptides in the hemoglobin of smokers undoubtedly indicates the multidirectional harmful effects of the aldehyde, which still requires better understanding [ 67 ].…”

Mass Spectrometry Analysis of DNA and Protein Adducts as Biomarkers in Human Exposure to Cigarette Smoking: Acrolein as an Example

“…We used the bottom-up proteomic strategy to identify and evaluate the physiologically relevant acrolein-modified peptides of human Hb after reducing them to stable protein adducts by high-resolution accurate MS. The Schiff base and FDP adducts of lysine and the Michael type of adducts at Cys, His, and Lys residues were identified …”

“…The Schiff base and FDP adducts of lysine and the Michael type of adducts at Cys, His, and Lys residues were identified. 98 3.2. Quantitation.…”

“…The results indicate that the levels of Michael-type adduct at α-Lys-16, α-His-50, and β-Lys-59 and the three ethylated peptides are significantly elevated in smokers (including oral cancer and healthy). 98 Cigarette smoke has been reported to result in the ethylation of DNA and Hb. 110,117−122 The levels of Michael adduct of acrolein-modified peptides and the ethylated peptides are strongly correlated, suggesting the connection of acrolein Hb adducts with smoking based on the areas under the receiver operating characteristic curve.…”

“…110,117−122 The levels of Michael adduct of acrolein-modified peptides and the ethylated peptides are strongly correlated, suggesting the connection of acrolein Hb adducts with smoking based on the areas under the receiver operating characteristic curve. 98…”