The purpose of this phase 1 SBIR grant is to develop a quantitative phosphopeptide biomarker assay for pulmonary arterial hypertension (PAH). The commercial product will consist of a kit of stable isotopically labeled internal standards of a selected set of phosphopeptides either up or down-regulated in PAH. The set of stable isotopically labeled peptides will be used in a parallel reaction monitoring (PRM) mass spectroscopy (MS) assay to quantify the level of target phosphoproteins in the buffy coat fraction of blood. We propose to determine if a subset of these phosphoproteins are differentially regulated in peripheral blood mononuclear cells (PBMCs) (buffy coat, which contains lymphocytes and platelets) of subjects with PAH compared to controls, to develop assays that can be used to determine how the levels of these phosphoproteins change over time, and to determine if these levels correlate with clinically important endpoints: i.e., mortality risk, hospitalization for PAH, and listing for lung transplant. We propose that a set of increased and decreased phosphoproteins in PBMCs will identify a subset of patients with a poor prognosis who are less likely to respond to currently approved therapies, and allow earlier interventions to modify the course of their disease and improve their outcomes. The chosen targets are based on a phosphoproteomic analysis of iPAH lung tissue compared to controls that Pulmokine performed in collaboration with the Rensselaer Center for Translational Research and the Yale University Proteomics core. Several dramatically increased and decreased phosphoproteins implicated in immune regulation, cell proliferation, and angiogenesis were identified. This feasibility study will develop a PRM-MS assay that can simultaneously quantify the phosphoproteins implicated in immune regulation, cell proliferation, and angiogenesis previously identified as highly regulated in iPAH and which will include several novel targets. The assay will be tested on buffy coat cell extracts from blood samples of iPAH and systemic sclerosis associated PAH (SSC APAH) with comparison to SSC without PH, and to normal controls. These samples will be obtained prospectively by Dr. Todd Bull, the director of the University of Colorado PH program. Phase 2 of the project will address regulatory requirements for kit commercialization, provide a longer validation study, and perform serial sampling of subjects with iPAH to correlate biomarker results with clinical course and prognosis.