1304-P: Quantification of Preproinsulin and Insulin Isoforms Derived from Upstream Open Reading Frames in Human Clinical Samples by Selected Reaction Monitoring Assay

We recently uncovered a novel human proinsulin containing a amino-acid Cα-peptide and upstream open reading frame (uORF) isoforms whose transcription is initiated from an alternative INS promoter upstream of the conventional INS promoter (Liu et al, 2021, Diabetes, PMID: 34649926) . Post-translational protease processing of preproinsulin is reported to be involved in the pathogenesis of diabetes. However, there are no sensitive and specific assays to quantify unprocessed preproinsulin and insulin originating from uORFs in clinical samples. Hence, we developed an LC-MS/MS based Selected Reaction Monitoring (SRM) assay to quantify the fragments of processed and unprocessed signal peptide in plasma, pancreatic islets, cerebrospinal fluid (CSF) , and brain. Trypsinization of insulin B-chain generates two peptides: peptide-B1 (pep-B1: FVNQHLCGSHLVEALYLVCGER) that requires cleavage by signal peptidase (SPase) , and peptide-B2 (GFFYTPK) that does not require SPase. As a result, the pep-B1 derived from preproinsulin cannot be detected if the signal peptide fails to be cleaved by SPase. Therefore, the pep-B1 could serve as a surrogate for preproinsulin since its quantity is correlated with the signal peptide cleavage efficiency. We also developed an SRM peptide panel to detect and measure insulin derived from uORF isoforms. Our SRM assay allows for measurement of insulin A- and B-chains separately in plasma samples, and levels highly correlate with ELISA after a glucose stimulus to secretion. We detected more unconventional uORF-derived insulin in CSF and cerebrum in comparison with plasma and islets. Furthermore, we found that uORF levels were significantly altered in middle temporal gyrus (MTG) of postmortem brains of Alzheimer Disease patients compared to control MTGs. The newly developed SRM assay could serve to determine preproinsulin and insulin uORF isoforms in clinical biofluid and tissue samples.