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

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.