Type 1 diabetes mellitus (T1DM) is a progressive autoimmune disease that attacks the β-cells of the endocrine pancreas, which results in a systemic loss of insulin and the ability to regulate blood glucose concentrations. It affects more than 1 million individuals in the US and the incidence in children is increasing at the alarming rate of 2.7-3.9% per year. By age 20, approximately 1 in 200 Caucasian American children will be diagnosed with T1DM. Currently the only treatment for T1DM is insulin replacement using injectable insulin/analogs, causing significant emotional and financial stressors on families, communities, and health care systems. Research continues in the areas of: (1) genetic and immunological factors that increase disease risk, (2) preventing disease development in at-risk individuals, (3) mechanisms that lead to the destruction of β-cells, (4) how hyper- and hypoglycemia lead to micro- and macrovascular disease, and (5) increasing adoption of technologies and interventions that reduce glycemic variability. While the triggers that lead to autoimmunity and the molecular transformations that lead to end-organ damage are poorly understood, many in vitro studies and mouse models have provided new directions for research. Unfortunately, the tools needed to translate new discoveries between labs or further into pre-clinical and clinical studies are often lacking.
More specifically, investigators must rely on immunoassays, such as ELISA or Western blotting, to test hypotheses involving protein expression or post-translational modification. Antibodies for use in immunoassays are expensive and often, even in commercial settings, are not specific for the analytes against which they were raised. In addition, immunoassays for proteins in human serum and plasma samples are inherently problematic due to endogenous interferences. To address these issues and in response to NIDDK RFA DK-17-019 we propose this project entitled, “Quantifying proteins in plasma to democratize personalized medicine for patients with type 1 diabetes,” during which we will develop, validate, and disseminate novel assays for use in biological and translational research that will help discover new pathways and therapeutic targets as well as enable researchers to test their hypotheses and evaluate the success of their interventions in human populations on the epidemiological scale.
Our laboratory has spent the last 12 years developing, calibrating, validating, and deploying novel mass spectrometric assays for proteins in human samples. In collaboration with laboratories around the world, we have demonstrated the ability to translate the fundamental principles of proteolysis and liquid chromatography- tandem mass spectrometry (LC-MS/MS) into assays that outperform immunoassays in translational research and in the care of patients. We will apply this experience to the study of T1DM in the following specific aims:
Specific Aim 1: Use data-independent acquisition strategies to multiplex targeted assay development of plasma proteins relevant to T1DM. Narrow-window data-independent analyses of pools of human samples and/or purified proteins will be used to identify peptides with good characteristics for LC-MS/MS assays: (1) detectable, (2) stable, (3) precise, and (4) linear.
Specific Aim 2: Develop enrichment strategies to quantify must-have low abundance analytes. There are many analytes that are important to the investigation of T1DM that are below the detection limits possible for simple trypsin digestion-LC-MS/MS assays. Included in this list are the five proteins listed in the RFA (i.e., insulin, c-peptide, somatostatin, glucagon, and glycated CD59). In order to quantify these proteins and other proteins important in T1DM research, we will develop enrichment strategies that include solid phase extraction and immunoaffinity enrichment of intact proteins or peptides after proteolysis.
Specific Aim 3: Validate multiplexed assays in plasma that are important in T1DM research. As described in guidelines from Clinical Chemistry and the Clinical Laboratory Standards Institute (e.g., C62-A), meaningful figures of merit will be generated, including analytical sensitivity (as measured by limits of detection and lower limit of quantification), specificity, accuracy, and precision.
Specific Aim 4: Disseminate assays and reagents to other laboratories. To widely distribute these assays to researchers and clinical laboratories, standard operating procedures and data from validation studies (in Skyline format) will be published online using Passport, Panorama Public, and Chorus. Additionally, hybridomas and cloned antibodies will be made available through the Developmental Studies Hybridoma Bank at the University of Iowa.