ABSTRACT Type 1 diabetes affects more than 1.25 million people in the United States and the annual incidence is increasing at an alarming rate of 3-4%. The emotional and financial burden of the disease is overwhelming and we currently have no way to predict or prevent new cases. As we gain a better understanding of the pathophysiological processes in the pancreas and the downstream effects of hyperglycemia (and periodic hypoglycemia during treatment), more robust biomarker assays are needed to improve the reproducibility of research findings and to translate those findings to clinical care. One technology that can provide robust, transferable assays for the measurement of proteins is liquid chromatography-tandem mass spectrometry. By directly detecting the analyte of interest, assays that use mass spectrometry detection can have better specificity than immunoassays and when paired with enrichment strategies, they can also be very sensitive. As we have demonstrated previously, it is straightforward to harmonize the results of mass spectrometric assays, which is significantly more difficult for immunoassays in general. This proposal aims to generate and validate novel transferable protein assays that harness the power of mass spectrometry. We aim to leverage a new method for the enrichment of extracellular vesicles and new de novo proteins for affinity enrichment, called minibinders, to help with sensitivity of the methods. Whenever possible, assays will be multiplexed and if antibodies are required for enrichment, they will be widely distributed through the Iowa Hybridoma Bank. Plasmids encoding minibinders will be deposited at Addgene. Chromatographic data from method development (particularly peptide selection, which will use narrow-window data-independent acquisition rather than relying on algorithms or data- dependent acquisition methods) as well as chromatographic data from method validation will be distributed via Panorama, along with detailed standard operating procedures. As requested in RFA DK-21-031, a portion of the assays produced will target glucagon, other fragments of proglucagon, proinsulin and its fragments, glycated soluble CD59, amylin, and the chromogranins. Our Target Prioritization Committee will help identify the most important proteins to add to this list and focus our development efforts.