A Novel high resolution MS platform for high-throughput screening of G protein-coupled receptors

用于高通量筛选 G 蛋白偶联受体的新型高分辨率 MS 平台

基本信息

批准号：
10636377
负责人：
Jon Jacobs
金额：
$ 34.23万
依托单位：
BATTELLE PACIFIC NORTHWEST LABORATORIES
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-15 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10636377
关键词：
Adaptor Signaling Protein Address Agonist Amino Acid Motifs Amino Acids Antibodies Biological Biological Assay Biology CXC chemokine receptor 3 CXCR3 gene Cell physiology Clinical Collection Complex Coupled Data Data Analyses Detection Development Dimensions Disease Dose Drug Screening Drug Targeting FDA approved Family G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Proteins Heterotrimeric GTP-Binding Proteins Inflammatory Ions Isomerism Ligands Mass Spectrum Analysis Membrane Modernization Pathway interactions Pattern Peptide Library Peptides Pharmaceutical Preparations Pharmacology Phosphopeptides Phosphorylated Peptide Phosphorylation Phosphorylation Site Phosphotransferases Play Process Protein Region Proteins Proteomics Reagent Reproducibility Research Resolution Role Sampling Signal Transduction Site Specificity Spectrometry Speed Structure System T cell regulation T-Cell Activation T-Lymphocyte Technology Testing Time Transducers Travel United States National Institutes of Health antagonist beta-arrestin cell preparation chemokine computerized data processing design drug development drug discovery experience high throughput screening improved informatics tool ion mobility mass spectrometer novel novel strategies overexpression phosphoproteomics programs receptor recruit response screening small molecule small molecule libraries ultra high resolution

Adaptor Signaling Protein Address Agonist Amino Acid Motifs Amino Acids Antibodies Biological Biological Assay Biology CXC chemokine receptor 3 CXCR3 gene Cell physiology Clinical Collection Complex Coupled Data Data Analyses Detection Development Dimensions Disease Dose Drug Screening Drug Targeting FDA approved Family G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Proteins Heterotrimeric GTP-Binding Proteins Inflammatory Ions Isomerism Ligands Mass Spectrum Analysis Membrane Modernization Pathway interactions Pattern Peptide Library Peptides Pharmaceutical Preparations Pharmacology Phosphopeptides Phosphorylated Peptide Phosphorylation Phosphorylation Site Phosphotransferases Play Process Protein Region Proteins Proteomics Reagent Reproducibility Research Resolution Role Sampling Signal Transduction Site Specificity Spectrometry Speed Structure System T cell regulation T-Cell Activation T-Lymphocyte Technology Testing Time Transducers Travel United States National Institutes of Health antagonist beta-arrestin cell preparation chemokine computerized data processing design drug development drug discovery experience high throughput screening improved informatics tool ion mobility mass spectrometer novel novel strategies overexpression phosphoproteomics programs receptor recruit response screening small molecule small molecule libraries ultra high resolution

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项目摘要

Summary: Mass spectrometry (MS) has proven invaluable in studying the mechanisms of cellular signaling as MS platforms can directly provide amino acid residue site-specific phosphorylation data compared to traditional antibody-based approaches. However, limitations exist in current MS approaches in generating confident site-specific phosphorylation quantification. This is particularly evident in complex multi-phosphorylated protein motifs, where the detection of isomeric multi-phosphorylated peptides easily overwhelms any prediction scoring approach that is simply based upon the fragmentation spectra. There are many biological examples of hyperphosphorylated regions, where they are associated with receptor/ligand interactions, including G-protein coupled receptors (GPCRs), membrane receptors that are the most common targets for FDA-approved drugs. For accurate site-specific quantification of protein hyperphosphorylation we propose a transdisciplinary approach using ultrahigh resolution Ion Mobility Separation (IMS) integrated with highly accurate and sensitive MS and MS/MS spectra to enable the confident characterization of hyperphosphorylated GPCR ensembles with greatly improved sensitivity, and speed. We will use multi-level Structures for Lossless Ion Manipulations (SLIM) technology (SLIM-Orbitrap platform) to fully characterize phosphorylation of GPCR/antagonist interactions utilizing CXCR3, which plays a central role in inflammatory diseases through its regulation of T cell function as an initial test case. We plan to first integrate ultrahigh resolution IMS with an advanced Orbitrap MS platform for unambiguous decoding of hyperphosphorylated sites, evaluate the SLIM-Orbitrap MS platform for resolving hyperphosphorylated protein regions, and finally, perform comprehensive site-specific phosphoproteomics for GPCRs through screening of activated T cells with dose-responses of chemokine and small-molecule CXCR3 biased agonists.

摘要：质谱法（MS）在研究细胞的机制方面已证明是无价的信号作为MS平台可以直接提供氨基酸残基特异性磷酸化与传统的基于抗体的方法相比，数据。但是，当前存在限制 MS在产生自信的位点特异性磷酸化定量方面的方法。这是在复杂的多磷酸化蛋白基序中特别明显，其中检测异构体多磷酸化肽很容易压倒任何预测评分方法仅基于碎裂光谱。有许多生物学例子高磷酸化区域，与受体/配体相互作用相关，包括G蛋白偶联受体（GPCR），最常见的膜受体 FDA批准的药物的靶标。为了准确对蛋白质的位点特异性定量高磷酸化我们提出了使用超高分辨率离子的跨学科方法与高度准确和敏感的MS和MS/MS光谱集成的移动性分离（IMS）为了使高磷酸化GPCR合奏的自信表征与提高灵敏度和速度。我们将使用多层结构进行无损离子操作（Slim）技术（Slim-Orbitrap平台），以充分表征使用CXCR3的GPCR/拮抗剂相互作用，在炎症中起着核心作用通过其对T细胞功能的调节作为初始测试案例的疾病。我们计划首先整合超高分辨率IMS具有高级Orbitrap MS平台，用于明确解码高磷酸化位点，评估用于解决的细长轨道MS平台高磷酸化的蛋白质区域，最后执行全面的位点特异性通过用剂量反应筛选活化的T细胞，用于GPCRS的磷酸蛋白质组学趋化因子和小分子CXCR3偏置激动剂。