Mass spectrometric immunoassay of intact insulin and related variants for population proteomics studies

Purpose: The purpose of the work presented herein was to develop a high-throughput assay for the quantification of human insulin in plasma samples while simultaneously detecting, with high mass accuracy, any additional variant forms of insulin that might be present in each sample. Experimental design: A mass spectrometric immunoassay (MSIA) was designed in which anti-human insulin antibodies were immobilized to commercially available mass spectrometric immunoassay pipette tips and used to capture insulin and related protein variants from human plasma. Results: Standard curves for insulin exhibited linearity (average R ² for three days of analysis=0.99) and assay concentration limits of detection and limits of quantification for insulin were found to be 1 and 15pM, respectively. Estimated coefficient of variations for inter-day experiments (n=3 days) were <8%. Simultaneously, the assay was shown to detect and identify insulin metabolites and synthetic insulin analogs (e.g. Lantus). Notably, insulin variants not known to exist in plasma were detected in diabetics. Conclusions and clinical relevance: This introductory study sets a foundation toward the screening of large populations to investigate insulin isoforms, isoform frequencies, and their quantification.