Identification of GltB-interacting proteins by CoIP-MS

To investigate whether GltB possess more function, Co-immunoprecipitation coupled with Mass Spectrometry (CoIP-MS) was used to isolate the putative GltB binding proteins.We used strains PAO1/p-gtrS-YFP and ΔgltB/p-gtrS-YFP to ensure the membrane protein GtrS of great interest is at high levels. A target GltB-specific antibody anti-GltB was used and ΔgltB/p-gtrS-YFP was set up as a negative control. The whole gel lane was divided into small fractions and in-gel digested for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Control sample was also analyzed in parallel to distinguish the background proteins.为探究GltB是否具备更多功能，本研究采用免疫共沉淀联合质谱（CoIP-MS）技术分离潜在的GltB结合蛋白。为确保我们重点关注的膜蛋白GtrS实现高表达，我们选用菌株PAO1/p-gtrS-YFP与ΔgltB/p-gtrS-YFP开展实验。实验使用靶向GltB的特异性抗体anti-GltB，并以ΔgltB/p-gtrS-YFP作为阴性对照。将整条凝胶泳道切割为若干小片段，经胶内酶解后进行液相色谱-串联质谱（LC-MS/MS）分析。同时平行分析对照样本，以区分背景干扰蛋白。