Introduction and Objective: Persistent proinsulin secretion and elevated proinsulin-to-C-peptide ratios have been reported in T1D. However, the potential of individual proinsulin proteoforms as biomarkers in T1D has not been evaluated due to the challenges associated with assay specificity. Herein, we present the first assessment of intact proinsulin, des-31,32 proinsulin, and des-64,65 proinsulin using a newly developed liquid chromatography-mass spectrometry (LC-MS)-based assay.Methods: The LC-MS assay utilized a cocktail of three monoclonal antibodies for enrichment, followed by LC-MS measurement. Serum samples from at-risk individuals (positive for two or more autoantibodies), new onset T1D subjects, and controls matched for age and sex (n=20 per group) were used for the initial assessment.Results: The LC-MS assay for measuring proinsulin proteoforms and C-peptide achieved a lower limit of quantification in the 1-5 pg/mL range, offering superior sensitivity and specificity than the Mercodia proinsulin assay. Across 80 samples, median levels were 915 pg/mL for C-peptide, 23.1 pg/mL for intact proinsulin, 46.2 pg/mL for des-31,32, and 4.6 pg/mL for des-64,65. The ratios of each of the three proinsulin proteoforms to C-peptide were significantly higher in T1D compared to controls, though no significant differences were observed between at-risk subjects and controls. Notably, the des-31,32-to-C-peptide ratio emerged as the most promising biomarker for differentiating T1D from controls. Moreover, the ratios of intact proinsulin, des-31,32, and des-64,65 to C-peptide showed poor correlation (R² ~0.5), suggesting they may serve as biomarkers for distinct etiologies of beta cell stress. Further assessment of the prognostic values of these markers is ongoing with an expanded sample set.Conclusion: All three proinsulin proteoforms displays promising and distinctive features in differentiating T1D subjects versus matched controls.
Q. Shen: None. W. Cao: None. T. Lin: None. C. Evans-Molina: Advisory Panel; DiogenX. Research Support; Bristol-Myers Squibb Company, Lilly Diabetes. Advisory Panel; Isla Technologies, Neurodon. Research Support; Neurodon. E.K. Sims: Consultant; Sanofi. Speaker’s Bureau; Med Learning Group. Other Relationship; American Diabetes Association. J. Qu: None. W. Qian: None.
National Institutes of Health (U01DK137113)
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