Lysine post-translational modifications are important regulators of protein function. Proteomic and biochemical approaches have resulted in identification of several lysine modifications, including acetylation, crotonylation, and succinylation. Here, we developed an approach for surveying amide-bonded lysine modifications in the proteome of human tissues/cells based on the observation that many lysine modifications are amide-bonded and that the Salmonella enterica deacetylase, CobB, is an amidase. After the proteome of human tissues/cells was denatured and the non-covalently bonded metabolites were removed by acetone washes, and the amide-bonded modifiers were released by CobB and analyzed using liquid- and/or gas chromatography/mass spectrometry metabolomic analysis. This protocol, which required 3–4 days for completion, was used to qualitatively identify more than 40 documented and unreported lysine modifications from the human proteome and to quantitatively analyze dynamic changes in targeted amide-bonded lysine modifications. We developed a method that was capable of monitoring and quantifying amide-bonded lysine modifications in cells of different origins.

中文翻译：

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使用脱乙酰酶 CobB 对酰胺键合赖氨酸修饰进行全细胞调查

赖氨酸翻译后修饰是蛋白质功能的重要调节剂。蛋白质组学和生化方法已导致鉴定了几种赖氨酸修饰，包括乙酰化、巴豆酰化和琥珀酰化。在这里，我们基于观察到许多赖氨酸修饰是酰胺键合的并且肠沙门氏菌脱乙酰酶 CobB 是一种酰胺酶，开发了一种用于调查人体组织/细胞蛋白质组中酰胺键合赖氨酸修饰的方法。在人体组织/细胞的蛋白质组变性并通过丙酮洗涤去除非共价键合的代谢物后，通过 CobB 释放酰胺键合的改性剂并使用液相和/或气相色谱/质谱代谢组学分析进行分析。该协议需要 3-4 天才能完成，用于定性识别来自人类蛋白质组的 40 多种记录和未报告的赖氨酸修饰，并定量分析靶向酰胺键合赖氨酸修饰的动态变化。我们开发了一种能够监测和量化不同来源细胞中酰胺键合赖氨酸修饰的方法。