Citation: XU Zhong-Hang, WU Yuan-Yu, LI Chun-Sheng, HE Cheng-Yan, FANG Xue-Dong. Selective Labeling and Proteomic Analysis of Free Thiols in Mitochondrial Proteins from Human Colon Carcinoma[J]. Chinese Journal of Analytical Chemistry, ;2023, 51(1): 84-92. doi: 10.19756/j.issn.0253-3820.221521 shu

Selective Labeling and Proteomic Analysis of Free Thiols in Mitochondrial Proteins from Human Colon Carcinoma

China-Japan Union Hospital, Jilin University, Changchun 130033, China

Corresponding author: HE Cheng-Yan, FANG Xue-Dong,
Received Date: 23 October 2022
Revised Date: 14 November 2022

Fund Project: Supported by the Jilin Province Science and Technology Department (Nos.20200404193YY, 20190201008JC).

The redox state of the thiol groups of protein cysteine residues is closely related to the local redox level of cells. When these thiol groups are oxidized or reduced, they can greatly affect protein structure, thereby modulating their biological functions and eventually affecting the biological processes and cell fate. In this study, a strategy aiming at selectively labeling the free thiol group of protein cysteine was proposed. In this method, N-ethylmaleimide (NEM), a thiol reactive reagent, was used to block the free thiol groups on the proteins prior to the routine sample processing in proteomics flow (Reduction of disulfide bond by dithiothreitol, alkylation blockage byiodoacetamide). Therefore, the original free thiol groups, as well as those generated from the reduction treatment by dithiothreitol, were blocked with two different thiol reactive reagents with different molecular weights, leading to specific identification of the original free thiol groups within proteins. By using this strategy, a proteomic investigation was performed on the free thiol groups of mitochondrial proteins in colorectal cancer tissues. A total of 1549 mitochondrial proteins were identified, including protein disulfide-isomerase A3, peroxiredoxin-1, mitochondrial NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial inner membrane protein, mitochondrial acetyl-CoA acyltransferase, malate dehydrogenase,calnexin, mitochondrial aspartate aminotransferase, mitochondrial succinate dehydrogenase [ubiquinone] iron-sulfur subunit, etc. Specially, 348 peptides containing free sulfhydryl groups were identified, belonging to 253 proteins. The proteomics data of the mitochondrial proteins as well as the peptides containing free thiols in colon cancer tissues could provide new ideas for further investigation on the redox targets as well as novel biomarkers in mitochondrial proteins in colon cancer.
- Mitochondria,
- Colon cancer,
- Proteome,
- Free thiol,
- Redox
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