大肠癌线粒体蛋白质自由巯基的选择性标记与组学分析  

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

作　　者：徐仲航 吴元玉[1] 李春生[1] 何成彦[1] 房学东[1] XU Zhong-Hang;WU Yuan-Yu;LI Chun-Sheng;HE Cheng-Yan;FANG Xue-Dong(China-Japan Union Hospital,Jilin University,Changchun 130033,China)

机构地区：[1]吉林大学中日联谊医院,长春130033

出　　处：《分析化学》2023年第1期84-92,共9页Chinese Journal of Analytical Chemistry

基　　金：吉林省科技厅项目(Nos.20200404193YY,20190201008JC)资助。

摘　　要：蛋白质半胱氨酸残基侧链巯基的氧化还原状态与细胞局部氧化还原水平密切相关,这些巯基被氧化或还原时,可极大地影响其结构,进而改变和调节其生物学功能,并对生物过程和细胞的命运产生决定性影响。本研究提出了一种选择性标记蛋白质半胱氨酸自由巯基的策略,即在常规的蛋白质组样本处理步骤(二硫苏糖醇还原二硫键、碘乙酰胺烷基化封闭)之前,使用具有巯基反应活性的N-乙基马来酰亚胺对蛋白质上的自由巯基预先进行封闭修饰,使蛋白质原有的自由巯基与经二硫苏糖醇还原而生成的自由巯基分别被具有不同分子量的巯基活性试剂封闭修饰,达到对蛋白质自由巯基特异性识别和表征的目的。利用以上策略,本研究对大肠癌组织中的线粒体蛋白自由巯基进行分析,共鉴定出1549种线粒体蛋白,包括蛋白质二硫键异构酶A3、过氧化物还原酶-1、线粒体NADH脱氢酶黄素蛋白2、线粒体内膜蛋白、线粒体乙酰CoA酰基转移酶、苹果酸脱氢酶、钙联蛋白、线粒体门冬氨酸氨基转移酶、线粒体琥珀酸脱氢酶[辅酶Q]铁硫亚基等;通过分析组学数据,共鉴定出348条含有自由巯基的肽段,归属于253种蛋白质。本研究对大肠癌组织中的线粒体蛋白和其中含有自由巯基肽段的组学研究结果进行了分析,可为进一步研究线粒体蛋白氧化还原作用靶点、精准发现大肠癌肿瘤线粒体蛋白标志物提供新的思路。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.

关 键 词：线粒体 大肠癌 蛋白质组 自由巯基 氧化还原 

分 类 号：R735.34[医药卫生—肿瘤]