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

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特别明显，其中检测 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。

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.