Soluble protein markers of T1D progression.

描述(由申请人提供)： 根据目前的遗传和自身抗体检测，预测1型糖尿病(T1D)进展的方法缺乏敏感性和特异性。在这份R21提案中，我们描述了基于蛋白质组学的研究方法，旨在识别和检测β细胞破坏和主动抗胰岛免疫的特定标记物。这项建议的总体战略是继续努力定位组织特异性、组织限制性和疾病诱导的人类胰岛基因产物。根据以前的工作，我们有一张人类胰岛组织限制性转录本的图谱。在与蛋白质质谱学专家(卡普托斯博士的团队)的合作下，我们现在建议利用这一独特的信息来加强对特定应用的可溶性人类胰岛蛋白质的研究。我们认为，对纯化的人胰岛和体外培养的人胰岛蛋白质组的研究，将有助于鉴定组织限制性和/或疾病相关蛋白，其中一些将是新生的1型糖尿病和/或其进展的潜在有用的血清标志物。我们假设，其中一些标志物将代表β细胞破坏之前和/或驱动β细胞破坏的炎症过程的副产品，而与其他标志物一样，代表胰岛组织特异性或高度限制性的蛋白质，将提供β细胞损伤的证据或与β细胞团相关。我们认为，由于β细胞的破坏，其中一些标志物可能以反映不完全翻译后修饰的形式存在于血清中。借鉴心肌缺血后心肌特异性蛋白释放的模型，在特定目的一，我们提出研究人T1D大鼠BB模型的血清蛋白，目的是确定胰岛特异性蛋白是否在β细胞被破坏后释放到循环中。利用信息学工具和传统的蛋白质测序方法，我们将识别与β细胞破坏和糖尿病相关的血清蛋白质和多肽。在特定的目标二中，我们将研究纯化的人胰岛的蛋白质组，目的是鉴定最普遍的胰岛组织特异性蛋白，特别是鉴定它们的翻译后修饰水平。同时，我们将开发一种基于SELDI-TOF的免疫分析芯片，由识别β细胞特异性抗体、神经内分泌组织特异性抗体和/或胰岛炎症相关蛋白标志物组成。标记的选择是基于我们对丰富的胰岛转录本的了解和从我们的胰岛蛋白质组特征中获得的信息。在确定了胰岛芯片的性能特征后，我们将回到大鼠模型，回顾寻找血清中的这些标记物，并确定它们与β细胞损伤和糖尿病的关系。我们已经召集了一个跨学科的研究团队，共同的目标是开发破坏胰腺内分泌β细胞的标记物。

DESCRIPTION (provided by applicant): Prediction methods for the progression of type 1 diabetes (T1D), based on current genetic and autoantibody testing, lack sensitivity and specificity. In this R21 proposal we describe proteomics based methods of study aimed at the identification and detection of specific markers of beta cell destruction and active anti islet immunity. The overall strategy of this proposal is to continue our efforts in mapping tissue specific, tissue restricted, and disease induced gene products of human islets. From previous work we have at our disposal a map of human islet tissue restricted transcripts. In collaboration with experts in mass spectroscopy of proteins (Dr. Caputos' group), we now propose to take advantage of this unique information to enhance studies of soluble human islet proteins with specific applications in mind. We propose that the study of the proteome of purified human islets and those cultured in interferon alpha ex vivo, will lead to the identification of tissue restricted and/or disease associated proteins, some of which will represent potentially useful serum markers of nascent type 1 diabetes and/or its progression. We hypothesize that some of these markers will represent by-products of inflammatory processes that precede and/or drive beta cell destruction, where as other markers, representing proteins either specific to or highly restricted to islet tissue, will provide evidence of beta cell damage or correlate with beta cell mass. We propose that due to beta cell destruction, some of these markers may be present in serum in forms that reflect incomplete post-translational modification. Drawing from the model of release of cardiac muscle specific proteins following myocardial ischemia, in Specific Aim One, we propose to study serum proteins in the rat BB model of human T1D with the goal of determining whether islet specific proteins are released into circulation following the destruction of beta cells. Using informatics tools and traditional protein sequencing methods we will identify serum proteins and peptides that correlate with beta cell destruction and diabetes. In Specific aim two, we will study the proteome of purified human islets, with the goals of identifying the most prevalent islet tissue specific proteins, with a particular focus on identifying their levels of post translational modification. In parallel, we will develop a SELDI-TOF based immunoassay chip, composed of antibodies recognizing beta cell specific, neuroendocrine tissue specific and/or islet inflammation-associated protein markers. The choice of markers is based on our knowledge of abundant islet transcripts and information we obtain from our islet proteome characterization. Having established the performance characteristics of the Islet chip, we will return to the rat model and retrospectively search for these markers in serum and determine their association with beta cell damage and diabetes. We have drawn together an interdisciplinary research team with the common goal of developing of markers of the destruction of beta cells of the endocrine pancreas.