2242-P: A Novel Method Using LC-MS/MS to Quantify Proinsulin in Human Plasma

Introduction and Objective: Plasma proinsulin is used to detect insulinomas and has been proposed as a marker of beta-cell function, particularly when used as a ratio with C-peptide. Immunoassays remain the primary method for measuring proinsulin despite inherent limitations. We aimed to develop and validate a novel assay that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the sensitive and specific measurement of intact proinsulin and its partially processed forms. Methods: Sample preparation consists of protein precipitation, proteolysis with Glu-C, peptide immunoenrichment, and LC-MS/MS analysis of two surrogate peptides. RGFFYTPKTRREAE (RGF) spans the cleavage site for des-31,32-proinsulin and GSLQKRGIVE (GSLQ) spans the site for des-64,65-proinsulin. As a result, RGF quantifies intact and des-64,65-proinsulin and GSLQ quantifies intact and des-31,32-proinsulin. Calibration was performed using a purified protein calibration material (characterized for purity and concentration). Method comparison was made using two commercially available immunoassays (i.e., ALPCO and TECO). Results: The assay demonstrated good precision and linearity, with sensitivity down to 2.8 pM for RGF and 8.8 pM for GSLQ. Concentrations of the GSLQ peptide increase during childhood, suggesting that concentrations of des-31,32-proinsulin increase from birth through puberty, while the concentration of intact proinsulin remains stable. Method comparison with the TECO immunoassay indicated excellent agreement with the RGF peptide (R=0.99), suggesting specific detection of intact proinsulin. Method comparison with the ALPCO immunoassay and multivariable linear regression suggested significant interference from des-31,32-proinsulin and insulin. Conclusion: Our validated high-throughput assay is robust and represents a new tool for advancing studies of beta-cell function. A detailed standard operating procedure, well-characterized calibration materials, and monoclonal antibodies are available to facilitate adoption by other laboratories. Disclosure S. Shijo: None. E. Goonatilleke: None. L. Barahona Carrillo: None. K.L. Forrest: None. J.O. Becker: None. M. MacCoss: Consultant; Current; Thermo Fisher Scientific, Alnylam Pharmaceuticals, Inc. Advisory Panel; Current; Talus Biosciences, Profound Therapeutics. Research Support; Current; Waters, Sciex, Bruker, Agilent, Eisai Co., Ltd., Novartis Pharmaceuticals Corporation. H. Huynh: None. A.N. Hoofnagle: Research Support; Current; Waters, Inc. Funding National Institutes of Health (1U01DK137097)