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

点击翻译按钮获取中文摘要

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.