Characterization of protein adducts formed by toxic alkaloids by nano-scale liquid chromatography with mass
Chi-Hsien Chou1, Lea-Yea Chuang, Wei-Lung Tseng
1Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
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.
Insights
Betel quid chewing links to cancer. This study identifies new protein adducts from arecoline and arecaidine using mass spectrometry, aiding in discovering biomarkers for betel nut exposure.
Area of Science:
- Toxicology
- Biochemistry
- Analytical Chemistry
Background:
- Betel quid chewing is linked to cytotoxicity, genotoxicity, and carcinogenicity, including oral cancer and liver diseases.
- Arecoline and arecaidine from areca nut are potential biomarkers for betel quid use.
- Identifying protein adducts can reveal toxic mechanisms and exposure biomarkers.
Purpose of the Study:
- To develop a mass spectrometry (MS)-based method for detecting arecoline- and arecaidine-protein adducts.
- To establish a screening platform for novel protein adducts formed by arecoline and arecaidine.
- To identify potential new biomarkers of betel nut exposure.
Main Methods:
- Used bovine serum albumin to predict protein binding sites for arecoline/arecaidine.
- Treated cells with arecoline to identify adducts after metabolic processing.
- Analyzed human plasma samples using nano-scale liquid chromatography-MS (LTQ Orbitrap) after tryptic digestion.
Main Results:
- Confirmed cysteine as the predominant amino acid in protein adduct formation.
- Identified new protein adducts formed by arecoline and arecaidine.
- Demonstrated a method for modeling long-term exposure in human plasma.
Conclusions:
- Developed a novel screening platform for identifying arecoline- and arecaidine-protein adducts.
- Cysteine adducts are key indicators of exposure.
- This strategy can help discover new biomarkers for betel nut exposure and related toxicities.
