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.
