Robust Mass Spectrometric Protein/Peptide Assays for Type 1 Diabetes Clinical Applications

项目概要/摘要： 1型糖尿病（T1 D）是一种毁灭性的疾病，经常发生在儿童和年轻人中， 自身免疫介导的胰腺β细胞损失。监测疾病进展一直具有挑战性 和临床干预的有效性。因此，仍然迫切需要高度可靠的测定，其定量 蛋白质或肽激素（例如，胰岛素、胰高血糖素）和它们的特异性同种型（或蛋白质型）作为 内分泌和外分泌功能。这种分析将在促进有效监测 T1 D发作之前或之后的疾病进展或新型临床干预的疗效。最新 临床测定几乎完全依赖于抗体或其它亲和试剂的使用。但具体 亲和试剂的特异性通常是未知的或难以表征。靶向质谱（MS） 提出了一种有前途的替代免疫测定。因此，本申请的总体目标是 开发可靠的、蛋白质组特异性的和多重靶向的MS分析方法，用于以下蛋白质/肽分析物的列表： T1 D的重要性具体而言，我们的目标是开发用于以下组的多重靶向MS测定： 作为内分泌和外分泌功能标志物的靶点：胰岛素、胰高血糖素、胰淀素、嗜铬粒蛋白、生长抑素， 激素原同种型（例如，胰岛素原，胰高血糖素原），杂合胰岛素肽，胰蛋白酶原，糖化CD 59， 以及T1 D研究界感兴趣的其他标志物。为了便于充分验证稳健性 和可转移性，总体目标将通过以下方面的合作努力实现： 两个独立的目标MS实验室，并通过多实验室测定验证工作。具体而言，目标1将是 专注于建立最佳的检测配置，以确保检测血清中的内源性分析物 T1 D中特定蛋白质型或感兴趣肽的样本。目标2将集中于分析优化， 在重现性、稳定性、选择性、线性和限度方面的完整测定表征 量化还将进行实验室间试验方案转移和试验表征。目标3将 通过多实验室验证试验，通过分析 相同队列的临床血清样品和针对选定的免疫测定的基准 分析物如胰岛素和C肽。总之，该项目将建立高度可靠和易于转移的 多重靶向MS测定用于许多难以测量的T1 D标志物。预计这些分析将使 对监测胰腺内分泌/外分泌功能，疾病进展， 以及T1 D研究中临床干预的有效性。

Project Summary/Abstract: Type 1 diabetes (T1D) is a devastating disease often occurring in children and young adults resulting from the autoimmune-mediated loss of pancreatic β-cells. It has been challenging for monitoring the disease progression and the efficacy of clinical interventions. Therefore, a critical need remains for highly reliable assays that quantify proteins or peptide hormones (e.g., insulin, glucagon) and their specific isoforms (or proteoforms) as markers of endocrine and exocrine function. Such assays will play an important role in facilitating effective monitoring of disease progression or efficacy of novel clinical interventions prior to or following the onset of T1D. Most current clinical assays depend on the use of antibodies or other affinity reagents almost exclusively. However, the exact specificity of affinity reagents is often unknown or difficult to characterize. Targeted mass spectrometry (MS) presents a promising alternative to immunoassays. Therefore, the overall objective of this application is to develop reliable, proteoform-specific, and multiplex targeted MS assays for a list of protein/peptide analytes of significance in T1D. Specifically, we aim to develop multiplex targeted MS assays for the following panel of targets as markers of endocrine and exocrine function: insulin, glucagon, amylin, chromogranin, somatostatin, prohormone isoforms (e.g., proinsulin, proglucagon), hybrid insulin peptides, trypsinogen, glycated CD59, as well as other markers of interest to the T1D research community. To facilitate full validation of the robustness and transferability of the assays, the overall objective will be accomplished through the collaborative efforts of two independent targeted MS labs and through a multi-lab assay validation effort. Specifically, Aim 1 will be focused on establishing optimal assay configurations for confident detection of endogenous analytes in serum samples for specific proteoforms or peptides of interest in T1D. Aim 2 will be centered on assay optimization and full assay characterization in the aspects of reproducibility, stability, selectivity, linearity, and limit of quantification. Inter-lab assay protocol transfer and assay characterization will also be pursued. Aim 3 will demonstrate the robustness and utility of the assays through multi-lab validation of the assays by analyzing the same cohort of clinical serum samples and benchmarking against well-established immunoassays for selected analytes such as insulin and c-peptide. Together, the project will establish highly reliable and easy-to-transfer multiplex targeted MS assays for many difficult to measure T1D markers. These assays are expected to make a significant contribution to the monitoring of pancreatic endocrine/exocrine functions, the disease progression, as well as the efficacy of clinical interventions in T1D research.