LC-MS/MS analysis identified CgCrzA phosphorylation sites in the wild-type and ΔCgmkk1 mutant expressing CgCrzA.zip

To identify the phosphorylation sites of CgCrzA, total proteins were extracted from the WT/CgCrzA-GFP and D<i>Cgmkk1</i>/CgCrzA-GFP transformants, and subjected to purification steps described previously. Purified proteins were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gel bands corresponding to CgCrzA were excised and analyzed using a Thermo Q-Exactive high-resolution mass spectrometer (Thermo Scientific, Waltham, MA). Phosphorylated sites were identified by MS/MS spectra data with Mascot Distiller (Matrix Science; version 2.4).

为鉴定CgCrzA的磷酸化位点，本研究从野生型（WT，Wild Type）/CgCrzA-绿色荧光蛋白（GFP）以及ΔCgmkk1/CgCrzA-GFP转化菌株中提取总蛋白，并按照此前报道的实验步骤完成蛋白纯化。纯化所得蛋白通过10%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行分离，切取对应CgCrzA蛋白的凝胶条带后，使用赛默飞Q-Exactive高分辨率质谱仪（赛默飞世尔科技，美国马萨诸塞州沃尔瑟姆市）进行分析。最终通过Mascot Distiller软件（Matrix Science；版本2.4）解析质谱二级（MS/MS）谱图数据，完成磷酸化位点的鉴定。