A Quantitative Chemical Proteomics Approach for Site-specific Stoichiometry Analysis of Ubiquitination
A Quantitative Chemical Proteomics Approach for Site-specific Stoichiometry Analysis of Ubiquitination
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DOI：
10.1002/anie.201810569
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发表时间：
2019-01-08
影响因子：
16.6
通讯作者：
Chen, Yue
中科院分区：
文献类型：
--
作者：
Li, Yunan;Evers, Jonathan;Chen, Yue
翻译后修饰的化学计量分析是一种对修饰的部分丰度进行绝对量化的新兴策略。在此，报道了一种用于泛素化化学计量分析的定量化学蛋白质组学工作流程，称为泛素化的同位素平衡定量(IBAQ-Ub)。该策略利用了一种新的胺反应化学标签(AcGG-NHS)，该标签在结构上与胰酶切割后修饰赖氨酸上泛素的GG残基同源，因此能够从胰酶消化和二次稳定同位素标记后的泛素化和未修饰赖氨酸残基生成结构相同的多肽。该策略非常稳健、灵敏和准确，使用蛋白质标准或复杂的细胞裂解物具有广泛的动态范围。因此，这项工作为泛素化信号通路的定量化学计量分析提供了一种有效的化学蛋白质组学工具。
Stoichiometric analysis of post-translational modifications is an emerging strategy for absolute quantification of the fractional abundance of the modification. Herein, a quantitative chemical proteomic workflow for stoichiometric analysis of ubiquitination is reported, named isotopically balanced quantification of ubiquitination (IBAQ-Ub). The strategy utilizes a new amine-reactive chemical tag (AcGG-NHS) that is structurally homologous to the GG remnant of ubiquitin on modified lysine after trypsin cleavage and therefore enables the generation of structurally identical peptides from ubiquitinated and unmodified lysine residues following trypsin digestion and secondary stable isotopic labeling. The strategy is highly robust, sensitive, and accurate with a wide dynamic range using either protein standards or complex cell lysates. Thus, this work provides an efficient chemical proteomics tool for quantitative stoichiometric analysis of ubiquitination signaling pathways.