PNNL

Abstract

Accurately measuring circulating proinsulin proteoforms is crucial for clinical investigation of diabetes and related diseases, but was previously not feasible owing to limited assay specificity/sensitivity. Here we devised a highly sensitive LC-MS-based strategy to quantify intact proinsulin, des-31,32 and des-64,65 proinsulin, and C-peptide in circulation. The method involves:i)quantitative, robust affinity capture using an optimized antibody cocktail, eliminating the severe quantitative bias across multiple proteomes typically introduced when using a single antibody; ii)Lys-C digestion producing unique signature peptides for each proteoform, and iii)trapping-nano-LC coupled with FAIMS/dCV-MS for an ultra-sensitive analysis. The selective trapping/delivery ensured sensitive/selective analysis of the targets while achieving excellent analytical robustness that is critical for clinical assays, and the FAIMS/dCV substantially reduces baseline noise/interferences, further enhancing S/N. The assay achieved exceptional sensitivity, with serum LOQs of 1.7, 2.3, and 3.6 pg/mL respectively for proinsulin, des-31,32 and des-64,65, representing the first assay capable of sensitively quantifying these major circulating proinsulin proteoforms. We applied this assay to a cohort of 80 subjects(prediabetes and new-onset type-1-diabetes with respective age/sex/BMI-matched controls, N=20/group), enabling the first accurate profiling of proinsulin proteoforms in clinical groups. The assay results demonstrated a clear separation of healthy and new-onset T1D groups that a parallel total-proinsulin ELISA assay fails to capture. Furthermore, distinct expression patterns in relative abundance ratios among proteoforms were observed across clinical groups. This assay may provide valuable insights into the ß-cell functions and the onset/progression of diabetes and other associated conditions. Moreover, the strategy is broadly applicable to targeted measurement of other biomarker proteoforms.