Mass Spectrometry platform for Oxidative Biomarker Discovery in Type 1 Diabetes

描述（由申请人提供）：1型糖尿病约占所有糖尿病病例的10%，其发病率在全球范围内持续增加。虽然新的治疗策略促进了预后的改善，但长期微血管并发症仍然是1型糖尿病患者发病和死亡的主要原因。心血管糖尿病自主神经病变（DAN）具有致残性临床后果，并可能通过改变电稳定性和损害心肌血流而促进恶性心律失常。人们认识到需要新的基于机制的生物标志物，以准确预测DAN并发症易发个体中激活的通路。本提案是对申请请求（RFA-DK-06-004）的回应，并直接解决了我们诊断能力中的这一关键差距。该提案的总体目标是使用基于质谱（MS）的方法开发和验证新的生物标志物，然后使用基于微阵列的蛋白质组学平台结合MS来阐明导致1型DAN的途径。该提案的一个主要焦点将涉及基于微阵列的蛋白质组学平台的开发，该平台可以很容易地转移到高通量临床应用中。利用高灵敏度和特异性的气相色谱质谱，我们最近表征和验证了冠心病和糖尿病患者血浆中氧化应激的特异性标志物。这些标记作为特定氧化途径的分子指纹。我们还开发了一个基于微阵列的蛋白质组学平台，可以准确地识别和筛选来自血浆蛋白质组的修饰蛋白。我们预测，使用我们的质谱方法，我们将能够识别血浆蛋白的差异氧化，从而独特地识别易患糖尿病损伤的患者亚群。我们假设新的氧化修饰蛋白生物标志物将预测糖尿病患者DAN的表型表达和该患者亚组对氧化还原调节治疗的临床反应。该提案的具体目的是：目的1：确定预测1型糖尿病患者DAN存在的氧化生物标志物。目的2：在接受联合抗氧化治疗或安慰剂治疗的已确定DAN患者队列中确定氧化生物标志物概况。目的3：确定生物标志物谱的改变是否可以预测DAN的功能测量。总的来说，这些使用新技术和系统方法的研究将提供对DAN发病机制的见解，并确定新的风险分层生物标志物，为未来大规模的前瞻性研究奠定基础。

DESCRIPTION (provided by applicant): Type 1 diabetes accounts for about 10% of all cases of diabetes and its incidence continues to increase worldwide. While newer treatment strategies have facilitated improved outcomes, the long-term microvascular complications remain the major cause of morbidity and mortality in patients with type 1 diabetes. Cardiovascular diabetic autonomic neuropathy (DAN) has disabling clinical consequences and may facilitate malignant arrhythmias by altering electrical stability and impairing myocardial blood flow. There is a recognized need for novel mechanism-based biomarkers that accurately predict pathways activated in the complication-prone individual with DAN. This proposal is in response to a request of application (RFA-DK-06-004) and directly addresses this critical gap in our diagnostic capability. The overall goals of this proposal are to develop and validate novel biomarkers using a mass spectrometry (MS) based approach followed by a microarray-based proteomic platform combined with MS to elucidate pathways that contribute to type 1 DAN. A major focus of the proposal will involve development of a microarray-based proteomic platform that can readily be transferred to high throughput clinical applications. Using highly sensitive and specific gas chromatography MS we have recently characterized and validated specific markers of oxidative stress in plasma of humans with CAD and diabetes. These markers serve as molecular fingerprints for specific oxidation pathways. We have also developed a microarray based proteomic platform that can accurately identify and screen modified proteins from the plasma proteome. We predict that using our MS approach we will be able to identify differential oxidation of plasma proteins which uniquely identifies subsets of patients vulnerable for diabetic damage. We hypothesize that novel oxidatively modified protein biomarkers will predict phenotypic expression of DAN in diabetic patients and clinical responses to redox-modulating therapy in this patient subgroup. The specific aims of this proposal are: Aim 1: To determine oxidative biomarkers that predict presence of DAN in subjects with type 1 diabetes. Aim 2: To define oxidative biomarker profiles in a cohort of patients with established DAN treated with combination antioxidant therapy or placebo. Aim 3: To determine if alterations in biomarker profile are predictive of functional measures of DAN. Collectively, these studies using novel techniques and a systematic approach will provide insights into the pathogenesis of DAN and identify novel biomarkers for risk stratification, laying the groundwork for future large scale prospective studies.