Identification of phosphorylation sites of purified ORP8 from in vitro AMPK kinase assay

We investigated the potential role of AMPK in the phosphorylation of ORP8.In vitro kinase assay using purified ORP8 and kinase-active AMPK complex confirmed that ORP8 is the direct substrate of AMPK. Phosphorylated ORP8 from in vitro kinase assay was analyzed by HPLC-MS/MS.

本研究探究了腺苷酸活化蛋白激酶（AMP-activated protein kinase，AMPK）在氧固醇结合蛋白相关蛋白8（Oxysterol-binding protein-related protein 8，ORP8）磷酸化过程中的潜在作用。采用纯化的ORP8与激酶活性型AMPK复合物开展体外激酶实验，结果证实ORP8为AMPK的直接底物。针对体外激酶实验获取的磷酸化ORP8样品，通过高效液相色谱-串联质谱（High Performance Liquid Chromatography-Tandem Mass Spectrometry，HPLC-MS/MS）完成了分析。