抗体非依赖的蛋白质泛素化修饰富集新方法及其在UBE2O调控底物研究中的应用
结题报告
批准号:
22004021
项目类别:
青年科学基金项目
资助金额:
24.0 万元
负责人:
孙明伟
依托单位:
学科分类:
分离与分析
结题年份:
2023
批准年份:
2020
项目状态:
已结题
项目参与者:
孙明伟
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中文摘要
深度覆盖的蛋白质泛素化修饰分析对研究泛素化蛋白质的功能至关重要。目前的方法存在样品回收率低、抗体富集选择性差和重现性低等问题,阻碍了蛋白质泛素化修饰的深度覆盖分析。本项目拟建立蛋白质泛素化修饰样品预处理新方法和泛素化修饰肽段的非抗体富集新方法:1)筛选生物相容性良好的离子液体作为裂解液,发展蛋白质泛素化修饰样品预处理方法,提高泛素化修饰肽段的回收率;2)使用标记试剂封闭蛋白质氨基,然后利用非特异性去泛素化酶去除底物泛素链,暴露泛素化修饰位点赖氨酸ε氨基;利用含有二硫键及生物素的试剂对暴露的赖氨酸ε氨基进行标记,先利用生物素进行富集,再打开二硫键释放含有修饰位点的肽段,建立抗体非依赖的蛋白质泛素化修饰富集新方法,提高蛋白质泛素化修饰分析的灵敏度、准确度和重现性;3)将新方法应用于解析UBE2O调控的泛素化修饰底物及修饰位点,为研究UBE2O异常与疾病发生发展的分子机制提供重要数据。
英文摘要
Comprehensive analysis of ubiquitinated substrates and ubiquitination sites is of great significance for studying the various functions of ubiquitination. Some aspects in current methods need to be improved, including low recovery, poor enrichment selectivity and low reproducibility, which hinder the in-depth protein ubiquitination analysis. Accounting for these problems, we will develop new methods about sample preparation and enrichment of ubiquitinated peptides in this project: 1) By screening for the biocompatibilities of ionic liquids, we will develop an ionic liquid based sample preparation method to improve the recovery of ubiquitination modification in the process of sample preparation; 2) After blocking free amino group by amino labeling reagent, the ubiquitin chain existed on substrate will be removed by non-specific deubiquitylating enzyme to expose the lysine ε amino group at the ubiquitination site. Then the lysine ε amino group at the ubiquitination site will be labeled by the amino labeling reagent that contains a biotin tag and a disulfide linkage. After affinity enrichment using biotin-streptavidin system, the peptides containing the ubiquitination sites will be released by opening disulfide linkage. The developed antibody-free enrichment method will improve the sensitivity, accuracy and reproducibility of protein ubiquitination analysis; 3) The methods will be used to systematically map the ubiquitination network regulated by UBE2O, providing important data for understanding the molecular mechanism of UBE2O abnormality in disease development.
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DOI:10.1016/j.aca.2023.340877
发表时间:2023-01
期刊:Analytica chimica acta
影响因子:6.2
作者:Mingwei Sun;Qing Zhang;Baofeng Zhao;Qiuling Huang;Wenfeng Wu;Peiyang Fan;Lihua Zhang;Xiaofei Zhang
通讯作者:Mingwei Sun;Qing Zhang;Baofeng Zhao;Qiuling Huang;Wenfeng Wu;Peiyang Fan;Lihua Zhang;Xiaofei Zhang
DOI:10.1186/s13578-022-00870-y
发表时间:2022-08-12
期刊:CELL AND BIOSCIENCE
影响因子:7.5
作者:Sun, Mingwei;Zhang, Xiaofei
通讯作者:Zhang, Xiaofei
国内基金
海外基金