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

• 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
• 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

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）已被证明在研究细胞凋亡机制方面是非常宝贵的。 作为MS平台的信号传导可以直接提供氨基酸残基位点特异性磷酸化 与传统的基于抗体的方法相比。然而，目前存在局限性。 MS方法在产生可信的位点特异性磷酸化定量。这是 在复杂的多磷酸化蛋白质基序中尤其明显，其中检测到 异构的多磷酸化肽容易克服任何预测评分方法， 仅仅是基于碎片光谱。有很多生物学上的例子 高度磷酸化的区域，在那里它们与受体/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批准的药物的目标。用于蛋白质的精确位点特异性定量 我们提出了一种跨学科的方法，使用高分辨率离子 迁移率分离（IMS）集成了高精度和高灵敏度的MS和MS/MS光谱 为了使过度磷酸化的GPCR集合的可靠表征具有极大的 提高了灵敏度和速度。我们将使用多能级结构进行无损离子操作 （SLIM）技术（SLIM-Orbitrap平台），以充分表征 利用CXCR 3的GPCR/拮抗剂相互作用，CXCR 3在炎症反应中起核心作用。 通过其调节T细胞功能的疾病作为初始测试案例。我们计划首先整合 具有先进Orbitrap MS平台的高分辨率IMS， 高度磷酸化位点，评估SLIM-Orbitrap MS平台用于解析 高度磷酸化的蛋白质区域，最后，进行全面的位点特异性 通过筛选具有以下剂量反应的活化T细胞的GPCR磷酸蛋白质组学 趋化因子和小分子CXCR 3偏向激动剂。