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

适用于 1 型糖尿病临床应用的稳健质谱蛋白质/肽检测

• 批准号: 10730900
• 负责人: Wei-Jun Qian
• 金额: $ 98.26万
• 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: 2023-07-21 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10730900
• 关键词: Affinity; Antibodies; Autoantibodies; Autoimmune; Benchmarking; Biological Assay; Blinded; Blood Glucose; C-Peptide; Child; Chromogranins; Clinical; Clinical Chemistry; Clinical Research; Collaborations; Communities; Coupled; Detection; Disease; Disease Progression; Endocrine; Failure; Fractionation; Genes; Glucagon; Goals; Human; Hybrids; Immunoassay; Indiana; Insulin; Insulin-Dependent Diabetes Mellitus; Intelligence; Intervention; Islets of Langerhans; Laboratories; Mass Spectrum Analysis; Measures; Mediating; Medicine; Methods; Monitor; Pathogenesis; Patients; Pediatric Hospitals; Peptides; Performance; Philadelphia; Play; Post-Translational Protein Processing; Proinsulin; Protein Isoforms; Proteins; Protocols documentation; Publications; RNA Splicing; Reagent; Reporting; Reproducibility; Research; Resolution; Role; Sampling; Serum; Somatostatin; Specificity; Standardization; Structure of beta Cell of islet; Trypsinogen; Universities; Validation; Variant; Work; assay development; clinical application; clinical efficacy; cohort; diabetes pathogenesis; experience; experimental study; glycation; high throughput screening; insulin dependent diabetes mellitus onset; interest; islet amyloid polypeptide; multiplex assay; nano; nanobodies; novel; peptide hormone; pressure; proglucagon; prohormone; serological marker; validation studies; young adult

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.

项目摘要/摘要： 1型糖尿病（T1D）是一种毁灭性疾病，通常发生在儿童和年轻人中 胰腺β细胞的自身免疫介导的损失。监测疾病进展一直是挑战 以及临床干预的效率。因此，对高度可靠的测定的关键需求仍然存在 蛋白质或胡椒恐怖（例如胰岛素，胰高血糖素）及其特定的同工型（或蛋白质成型）作为标记 内分泌和外分泌功能。此类测定将在促进有效监控的有效监控方面发挥重要作用 在T1D发作之前或之后，新型临床干预措施的疾病进展或有效性。最新 临床分析几乎完全取决于使用抗体或其他亲和力试剂的使用。但是，确切的 亲和力试剂的特异性通常是未知或难以表征的。目标质谱法（MS） 提出了免疫测定的承诺替代品。因此，此应用程序的总体目标是 开发可靠的，蛋白质的特异性和多重目标MS分析，以列出蛋白质/肽分析物的列表 T1D的重要性。特别是，我们旨在为以下面板开发多重目标MS分析 作为内分泌和外分泌功能标记的靶标：胰岛素，胰高血糖素，淀粉纤维，铬甘氨酸，生长抑素， 激素同工型（例如，促硫素，progulucagon），杂化胰岛素宠物，胰蛋白酶原，糖化CD59，AS 以及T1D研究社区感兴趣的其他标记。促进充分验证鲁棒性 和分析的可转让性，总体目标将通过合作的努力来实现 两个独立的目标MS实验室，并通过多LAB测定验证工作。具体来说，目标1将是 专注于建立最佳测定构型，以自信地检测串行中的内源分析物 特定蛋白质成型或t1d感兴趣的宠物的样品。 AIM 2将以测定优化为中心 在可重复性，稳定性，选择性，线性和极限方面的完整测定表征 数量。 LAB间测定协议转移和测定表征也将被追求。目标3意志 通过分析通过多LAB验证测定法的鲁棒性和实用性 相同的临床血清样品和针对精选的免疫测定的基准测试 胰岛素和C肽等分析物。该项目将共同建立高度可靠且易于转移 多重目标MS分析，许多难以测量T1D标记。这些测定有望做出 对监测胰腺内分泌/外分泌功能的重要贡献，疾病进展， 以及T1D研究中临床干预措施的效率。