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

使用定量磷酸蛋白质组学方法鉴定参与 SARS-CoV-2 进入的共受体和成分

基本信息

批准号：
10272278
负责人：
Tian Jin
金额：
$ 8万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10272278
关键词：
2019-nCoV Actins Affinity Chromatography Alkylation Binding Binding Proteins CCR5 gene COVID-19 pandemic CXCR4 gene Cell Nucleus Cells Chemicals Chemotactic Factors Chemotaxis Coronavirus Cytoskeleton Dictyostelium discoideum Digestion Folic Acid G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors Glycoproteins HIV HIV Envelope Protein gp120 Harvest High Pressure Liquid Chromatography Human Immobilization Knowledge Label Mass Spectrum Analysis Membrane Fusion Metals Methods Molecular Peptides Phagocytosis Pharmaceutical Preparations Phosphopeptides Phosphorylation Phosphorylation Site Proteins Reagent Receptor Protein-Tyrosine Kinases Signal Transduction Signaling Protein Surface Time Trypsin Viral Virus Virus Replication chemokine chemokine receptor folate-binding protein migration phosphoproteomics prevent receptor

项目摘要

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.

我们已经启动了使用定量磷酸化蛋白质组学方法鉴定参与 SARS-CoV-2 进入的辅助受体和成分的项目。我们开发了一种质谱方法来检测活细胞中蛋白质磷酸化在刺激后的变化。该方法结合了用于时间特异性定量的串联质量标签 (TMT) 化学标记和用于磷酸肽鉴定和磷酸化位点定量的多级 MS。具体来说，在刺激后的不同时间点收获细胞，裂解进行还原、烷基化和胰蛋白酶消化，每个时间点的肽均由单一TMT标记试剂特异性标记。将标记的肽混合并通过HPLC分离，然后使用固定化金属亲和层析富集磷酸肽，并进行质谱分析。第一个 MS 识别磷酸肽，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的叶酸受体（fAR1），它可以检测细菌表面的趋化剂叶酸和脂多糖，并调节盘基网柄菌中趋化和吞噬作用的肌动蛋白细胞骨架。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辅助受体（GPCR 或酪氨酸激酶受体）和信号蛋白。