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

摘要：质谱学(MS)已被证明在研究细胞的机制方面具有非常重要的价值。 作为MS平台的信号可以直接提供氨基酸残基位置特异的磷酸化 数据与传统的基于抗体的方法进行比较。然而，现行的法律制度存在着局限性。 MS方法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 同分异构体多磷酸化肽很容易压倒任何预测评分方法 只是基于碎裂光谱。有很多生物学上的例子 过度磷酸化区域，在那里它们与受体/配体相互作用有关， 包括G蛋白偶联受体(GPCRs)，膜受体是最常见的 FDA批准的药物的目标。为了准确地对蛋白质进行定点定量 我们提出了一种使用超高分辨率离子的跨学科方法 流动分离(IMS)与高精度和高灵敏度的MS和MS/MS光谱相结合 要使过度磷酸化的GPCR系综具有极大的可信度 提高了敏感度和速度。我们将使用多能级结构进行无损离子操纵 (SLIM)技术(SLIM-Orbitrap平台)充分表征磷酸化 利用CXCR3的GPCR/拮抗剂相互作用，在炎症中发挥核心作用 疾病通过其对T细胞功能的调节作为初步试验案例。我们计划首先整合 采用先进的Orbitrap MS平台的超高分辨率IMS，可明确解码 过度磷酸化的位点，评估SLIM-Orbitrap MS平台的解析 过度磷酸化的蛋白质区域，最后，执行全面的位点特异性 通过筛选具有剂量效应的活化T细胞研究GPCRs的磷酸蛋白质组学 趋化因子和小分子CXCR3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.