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酸蛋白质组学方法鉴定参与SARS-CoV-2进入的共受体和组分的项目。 我们开发了一种质谱方法来检测刺激后活细胞中蛋白质磷酸化的变化。 该方法结合了用于时间特异性定量的串联质量标签（TMT）化学标记和用于鉴定磷酸肽和定量磷酸化位点的多级MS。 具体地，在刺激后的不同时间点收获细胞，裂解用于还原、烷基化和胰蛋白酶消化，来自每个时间点的肽通过单一TMT标记试剂特异性标记。 标记的肽混合并通过HPLC分离，然后通过使用固定化金属亲和色谱富集磷酸肽，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，我们发现了长期寻求后的叶酸受体（fAR 1），检测化学引诱物叶酸和LPS在细菌表面和调节肌动蛋白细胞骨架的趋化性和吞噬在盘基网柄藻。 使用这种定量磷酸化蛋白质组学方法，我们将通过识别人类细胞中S蛋白触发的磷酸化增加来发现潜在的共受体（GPCR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.