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) 是一种毁灭性的疾病，通常发生在儿童和年轻人中，由糖尿病引起 自身免疫介导的胰腺 β 细胞损失对于监测疾病进展一直具有挑战性。 因此，仍然迫切需要高度可靠的定量分析方法。 蛋白质或肽激素（例如胰岛素、胰高血糖素）及其特定亚型（或蛋白质型）作为标记 此类测定将在促进有效监测方面发挥重要作用。 T1D 发病之前或之后的疾病进展或新型临床干预措施的疗效 最新。 临床测定几乎完全依赖于抗体或其他亲和试剂的使用。 亲和试剂的特异性通常未知或难以通过靶向质谱 (MS) 进行表征。 因此，该应用的总体目标是 为一系列蛋白质/肽分析物开发可靠的、蛋白质型特异性的、多重靶向的 MS 检测方法 具体来说，我们的目标是为以下组开发多重靶向 MS 检测。 作为内分泌和外分泌功能标志物的靶标：胰岛素、胰高血糖素、胰淀素、嗜铬素、生长抑素、 激素原异构体（例如胰岛素原、胰高血糖素原）、混合胰岛素肽、胰蛋白酶原、糖化 CD59，如 以及 T1D 研究界感兴趣的其他标记物，以促进稳健性的全面验证。 和检测的可转移性，总体目标将通过以下人员的共同努力来实现： 具体而言，目标 1 将通过两个独立的目标 MS 实验室并通过多实验室测定验证工作来实现。 专注于建立最佳检测配置，以可靠地检测血清中的内源分析物 目标 2 将集中于检测优化和 T1D 中感兴趣的特定蛋白型或肽的样品。 再现性、稳定性、选择性、线性和极限等方面的完整测定表征 还将追求实验室间检测方案转移和检测表征。 通过分析多个实验室的分析验证来证明该分析的稳健性和实用性 同一队列的临床血清样本，并与选定的成熟免疫测定进行基准比较 该项目将结合胰岛素和 C 肽等分析物建立高度可靠且易于转移的方法。 针对许多难以测量的 T1D 标记物进行多重靶向 MS 检测。 对监测胰腺内分泌/外分泌功能、疾病进展、 以及 T1D 研究中临床干预的功效。