Identification of co-receptor and components involved in the entry of SARS-CoV-2 using a quantitative phosphoproteomic approach

我们已经启动了使用定量磷蛋白质组学方法识别参与SARS-CoV-2进入的辅助受体和组件的项目。我们开发了一种质谱学方法来检测活细胞在刺激下蛋白质磷酸化的变化。该方法结合了串联质量标记(TMT)化学标记的时间特异性定量和多级MS的磷酸化多肽鉴定和磷酸化位点的定量。具体地说，在刺激的不同时间点收集细胞，裂解细胞进行还原、烷基化和胰酶消化，每个时间点的多肽被单一的TMT标记试剂特异性标记。将标记的多肽混合后用高效液相色谱分离，然后用固定化金属亲和层析富集磷酸多肽，并进行质谱分析。第一个MS鉴定磷酸肽，第二个MS确定磷酸化位点及其相对定量。利用这种方法，我们发现了人们期待已久的叶酸受体(FAR1)，它既能检测细菌表面的趋化剂叶酸，又能检测细菌表面的脂多糖，并调节盘基网柄菌的趋化和吞噬作用的肌动蛋白细胞骨架。利用这种定量的磷酸化蛋白质组学方法，我们将通过鉴定S蛋白在人类细胞中触发的磷酸化增加来发现潜在的辅助受体(gpr或酪氨酸激酶受体)和信号蛋白。

We have started the project Identification of co-receptor and components involved in the entry of SARS-CoV-2 using a quantitative phosphoproteomic approach. We developed a mass-spectrometry method to detect changes of protein phosphorylation in live cells upon stimulations. This method combines tandem mass tag (TMT) chemical labeling for time-specific quantification and multistage MS for identification of phosphopeptides and quantification of phosphorylation sites. Specifically, cells were harvested at different time points upon a stimulation, lysed for reduction, alkylation, and trypsin digestion, the peptides from each time point were specifically labeled by a single TMT labeling reagent. Labeled peptides were mixed and separated by HPLC, followed by enrichment of the phosphopeptides by using immobilized metal affinity chromatography, and subjected to mass spectrometry. The first MS identifies phosphorpeptides and the second MS determines the phosphorylation site and its relative quantification. Using this method, we discovered the long-sought-after folic acid receptor (fAR1) that detects both the chemoattractant folate and LPS on bacterial surface and regulates the actin cytoskeleton for both chemotaxis and phagocytosis in Dictyostelium discoideum. Using this quantitative phosphoproteomic approach, we will discover potential co-receptors (GPCR or Tyrosine kinase receptor) and signaling proteins by identifying S-protein-triggered phosphorylation increases in human cells.