Characterization of protein adducts formed by toxic alkaloids by nano-scale liquid chromatography with mass spectrometry

Abstract

Betel quid chewing is associated with cytotoxicity, genotoxicity and carcinogenicity in diseases such as oral cancer, liver cirrhosis, hepatocellular carcinoma and diabetes mellitus. Arecoline and arecaidine, which are the main alkaloids in the areca nut, are potential exposure biomarkers in habitual betel quid users. This study developed a method of detecting arecoline- and arecaidine-protein adducts by mass spectrometry (MS). First, bovine serum albumin was used to predict and confirm the binding sites of proteins modified by arecoline or arecaidine. Cells were then treated with arecoline to identify new protein adducts after cellular metabolic processing. Finally, human plasma was used to model long-term exposure to arecoline and arecaidine. Following isolation proteins were tryspin digested. The peptides afforded were separated and analyzed by nano-scale liquid chromatography with MS using an LTQ Orbitrap mass spectrometer. The experimental findings showed that cysteine is the predominant amino acid in protein adduct formation. The goal of this study was to establish a screening platform for identifying novel protein adducts that form covalent bonds with arecoline or arecaidine. Use of this strategy to survey new protein-toxic adducts may help to identify novel biomarkers of betel nut exposure.