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

项目摘要/摘要： 1型糖尿病(T1D)是一种破坏性疾病，常发生在儿童和年轻人中，原因是 自身免疫介导的胰腺β细胞丢失。监测疾病进展一直是个挑战。 以及临床干预的效果。因此，仍然迫切需要高度可靠的量化分析方法。 蛋白质或多肽激素(如胰岛素、高血糖素)及其特定的异构体(或蛋白异构体)作为 内分泌和外分泌功能。这种化验将在促进有效监测的过程中发挥重要作用 在T1D发病之前或之后，新的临床干预措施的疾病进展或疗效。最新版本 临床检测几乎完全依赖于抗体或其他亲和试剂的使用。然而，准确的 亲和试剂的特异性通常是未知的或难以表征的。靶向质谱学 为免疫分析提供了一种很有前途的替代方法。因此，此应用程序的总体目标是 为一系列蛋白质/多肽分析物开发可靠的、蛋白质形式特异的和多重靶向的MS分析方法 在T1D中的意义。具体地说，我们的目标是为以下小组开发多重靶向MS分析 内分泌和外分泌功能的标记物：胰岛素、胰高血糖素、胰淀素、嗜铬粒蛋白、生长抑素、 前激素亚型(如胰岛素原、胰高血糖素原)、混合胰岛素多肽、胰酶原、糖化CD59、AS 以及T1D研究界感兴趣的其他标记。以便于全面验证健壮性 和可转移性，总体目标将通过以下合作努力实现 两个独立的目标MS实验室，并通过多个实验室的分析验证工作。具体地说，目标1将是 专注于建立最佳的分析配置，以确保对血清中内源性分析物的可靠检测 T1D中感兴趣的特定蛋白形式或多肽的样本。目标2将集中在分析优化和 在重复性、稳定性、选择性、线性和检出限等方面的全面分析表征 量化。还将进行实验室间化验方案转让和化验鉴定。目标3将 通过多个实验室对分析结果进行分析，展示分析方法的稳健性和实用性 相同的临床血清样本队列，并与成熟的免疫分析方法进行比较 胰岛素和C-肽等分析物。总而言之，该项目将建立高度可靠和易于转移的 针对许多难以检测的T1D标志物进行多重靶向MS分析。这些化验有望使 对监测胰腺内/外分泌功能、疾病进展、 以及临床干预在T1D研究中的效果。

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.