Phosphopeptide mapping of plexiform lesions in pulmonary arterial hypertension

点击翻译按钮获取中文摘要

Pulmonary Arterial Hypertension (PAH) is a rare disorder of the pulmonary vasculature associated with high morbidity and mortality. The pathology of the disease consists of plexiform lesions of proliferating cells which obstruct blood flow through the pulmonary arterioles. There is a growing interest in the use of kinase inhibitors to address this underlying pathology. However, very little is actually understood about the regulation of kinases in PAH, and the phosphoproteome in PAH has not yet been studied. This project will determine the phosphopeptide profile of lung tissue from subjects with idiopathic pulmonary arterial hypertension (iPAH) compared to controls. Titanium dioxide chromatography will be used to enrich for phosphopeptides which will then be identified by mass spectroscopy. A bioinformatics approach will be used to predict the kinases and/or kinase families most likely to be responsible for these increased phosphoproteins. The NetworKIN and other algorithms will be used for this purpose. The predictions of NetworkKIN will be tested in a subset of kinases by incubating the selected recombinantly expressed kinases with peptide microarrays that duplicate the peptide sequences of the candidate phosphorylation sites. The findings of the proteomic analysis will be validated by isolating protein from plexiform lesions with laser capture microdissection. The proteins will be analyzed with a novel nanofluidic immunoassay that can detect very small quantities of protein. The results of this study could identify new therapeutic targets for the treatment of iPAH and thereby benefit patients and society.