Identification of SENP6 targets in HeLa cells by SENP6 SiRNA and MW shift analysis

The SUMO protease SENP6 disassembles SUMO chains and thus SENP6 depletion should lead to the extension of SUMO polymers on substrates. We have used a proteomic method to identify putative SENP6 substrates based on increased apparent molecular weight after SENP6 depletion. This method uses SDS-PAGE fractionation of 6His-SUMO2 purifications from cells and slicing of gels into multiple pieces before tryptic digestion and MS analysis. Gel slices are assigned an average Apparent MW based on molecular weight standards (run in a separate lane), and this information combined with protein intensities to give every protein an Apparent MW both in the presence of SENP6 and its ablation via SiRNA. The difference between the two values allows us to understand which proteins become more SUMOylated upon SENP6 knock-down.

SUMO蛋白酶SENP6（SUMO protease SENP6）可特异性解离SUMO链，因此SENP6耗竭会导致底物上的SUMO聚合物链延长。本研究基于SENP6耗竭后底物表观分子量（apparent molecular weight）升高的特征，采用蛋白质组学方法鉴定推定的SENP6底物。该方法的实验流程为：先对细胞中纯化得到的6His-SUMO2产物进行SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）分级分离，将凝胶切割为多个片段后，再依次进行胰蛋白酶消化与质谱（MS）分析。研究人员通过单独泳道的分子量标准品（molecular weight standards）为每个凝胶切片确定平均表观分子量，并结合蛋白丰度信息，分别计算出SENP6存在以及通过小干扰RNA（SiRNA）敲低SENP6时，每种蛋白的表观分子量。通过对比两组数值的差值，即可明确哪些蛋白质在SENP6敲低后发生了更为显著的SUMO化修饰。