Chemical Proteomic Profiling of Lysophosphatidic Acid-Binding Proteins
Chemical Proteomic Profiling of Lysophosphatidic Acid-Binding Proteins
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DOI：
10.1021/acs.analchem.9b04850
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发表时间：
2019-12-17
影响因子：
7.4
通讯作者：
Wang, Yinsheng
中科院分区：
文献类型：
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作者：
Dong, Xuejiao;Gao, Linfeng;Wang, Yinsheng
溶血磷脂酸（LPA）是一种内源性细胞信号分子，LPA信号通路的失调伴随着多种类型的癌症。在此，我们开发了一种化学蛋白质组学方法，用于在蛋白质组范围内鉴定lpa结合蛋白。该方法包括合成一种去硫代生物素偶联的LPA酰基磷酸探针，用于共价标记、富集和随后的LC-MS/MS鉴定LPA结合蛋白在蛋白质组水平。通过在两种不同探针浓度（10和100 μ M）下进行标记反应，并结合基于SILAC（细胞培养中氨基酸稳定同位素标记）的工作流程，我们在整个蛋白质组尺度上表征了这些蛋白的lpa结合能力，从而鉴定出HEK293T细胞中的86种候选lpa结合蛋白。此外，我们证实了其中两个蛋白，膜联蛋白A5和磷酸甘油酸激酶1，可以直接与LPA结合。我们共同开发了一种新的LPA探针，用于从整个人类蛋白质组中鉴定和表征LPA结合蛋白。该方法应适用于其他脂质结合蛋白的鉴定。
Lysophosphatidic acid (LPA) is an endogenous cell signaling molecule, and dysregulation of LPA signaling pathways is accompanied by several types of cancer. Herein, we developed a chemical proteomic method for the proteome-wide identification of LPA-binding proteins. The method involves the synthesis of a desthiobiotin-conjugated LPA acyl phosphate probe for the covalent labeling, enrichment, and subsequent LC-MS/MS identification of LPA-binding proteins at the proteome-wide level. By conducting labeling reactions at two different probe concentrations (10 and 100 mu M) in conjunction with an SILAC (stable isotope labeling by amino acids in cell culture)-based workflow, we characterized the LPA-binding capabilities of these proteins at the entire proteome scale, which led to the identification of 86 candidate LPA-binding proteins in HEK293T cells. Moreover, we validated that two of these proteins, annexin A5 and phosphoglycerate kinase 1, can bind directly with LPA. Together, we developed a novel LPA probe for the identification and characterizations of LPA-binding proteins from the entire human proteome. The method should be adaptable for the identification of other lipid-binding proteins.