Type 2 diabetes (T2D) is disease epidemic caused by a failure of beta cells to produce sufficient insulin to maintain euglycemia. As a consequence of obesity, insulin resistance develops that pressures the beta cell to produce elevated levels of insulin. Although beta cells have some capacity to compensate by increasing insulin production, a majority of individuals with insulin resistance develop beta cell failure and lose beta cell mass. Unfortunately the mechanism(s) leading to beta cell failure is not understood. We hypothesize that the fundamental cause of beta cell failure in T2D is an inability of the beta cell to fold pro-insulin within the endoplasmic reticulum (ER). To test this central cause of beta cell failure, we have brought together a team of established investigators to use state-of-the-art proteomics to identify the early changes within the secretory pathway that lead to beta cell demise. To accomplish this goal we propose three specific aims (SA). SA 1: To develop sensitive methods to quantify the percentage of folded versus misfolded pro-insulin in beta cells. SA 2: To establish procedures to quantitatively define the entire spectrum of proteins which interact with pro-insulin and insulin within the secretory pathway. SA 3: To devise organelle isolation and quantitative proteomics to map all the proteins within the ER, the Golgi network, and secretory granules of beta cells. To accomplish these aims we will function as a team to establish a novel platform to elucidate the proteomic organization of the beta cell secretory pathway to identify all the factors that contribute to insulin biogenesis. In the future, this proteomic approach will be applied to identify early prognostic markers that lead to beta cell failure and provide a foundation to evaluate new therapeutic modalities for T2D. RELEVANCE (See instructions): Type 2 diabetes (T2D) is an epidemic that results from an inability of beta cells to produce insulin. Recent findings support the notion that pro-insulin misfolding within the endoplasmic reticulum may be an initial event leading to beta cell failure. We propose to develop technologies to identify the protein network that promotes pro-insulin folding that may lead to future diagnostics and therapeutics for T2D.