Phosphopeptide mapping of plexiform lesions in pulmonary arterial hypertension

描述（由申请人提供）：肺动脉高压（PAH）是一种罕见的肺血管疾病，发病率和死亡率高。该疾病的病理包括增生细胞的丛状病变，其阻碍了肺小动脉的血液流动。有越来越多的兴趣使用激酶抑制剂来解决这一潜在的病理。然而，对PAH中激酶的调控实际上知之甚少，磷酸蛋白组在PAH中的研究尚未开展。本项目将测定特发性肺动脉高压（iPAH）患者与对照组肺组织的磷酸肽谱。将使用二氧化钛色谱法富集磷酸肽，然后用质谱法鉴定。生物信息学方法将用于预测激酶和/或激酶家族最有可能负责这些增加的磷酸化蛋白。NetworKIN和其他算法将用于此目的。NetworkKIN的预测将在一组激酶中进行测试，方法是将选择的重组表达的激酶与复制候选磷酸化位点肽序列的肽微阵列孵育。蛋白质组学分析的结果将通过激光捕获显微解剖从丛状病变中分离蛋白质来验证。这些蛋白质将用一种新型的纳米流体免疫分析法进行分析，这种方法可以检测到非常少量的蛋白质。本研究结果可以为iPAH的治疗找到新的治疗靶点，从而使患者和社会受益。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Pulmonary Arterial Hypertension (PAH) is a rare disorder of the pulmonary blood vessels associated with high morbidity and mortality. The purpose of this project is to identify proteins that may be responsible for the abnormal growth of cells that block the blood vessels and lead to the disease. This project is relevant to public health because it could lead to new treatments for PAH. (End of Abstract)