MS based approaches for diabetes biomarker discovery

描述(由申请人提供)：本提案的目标是应用新的亲和质谱学(AMS)技术来识别糖尿病前期或2型糖尿病的蛋白质生物标记物。所使用的方法将从复杂生物溶液中捕获微亲和蛋白与质谱学检测相结合。在第一阶段(R21)，我们将对提供的血浆和血细胞样本进行筛选，以确定候选蛋白质和蛋白质图谱，这些蛋白质和蛋白质谱可以适当区分样本来源和三组中的一组(正常、糖尿病前期和糖尿病；由口服葡萄糖耐量试验确定)。血浆和血细胞样本将用多克隆抗体和单抗衍生的亲和吸管进行分析，以进行特定的蛋白质靶标分析。此外，将通过亲水和疏水配体、金属螯合配体和其他广特异性亲和表面来分析蛋白质组。每一种配基表面都具有特定的亲和力特性，这些特性将导致捕获和纯化单个或整个蛋白质组。由于质量和/或数量差异而观察到的蛋白质变化，具有统计学意义，并根据其来源正确地对样本进行分组，将被视为潜在的生物标志物。在第二(验证)阶段(R33)，我们将通过使用高吞吐量并行机器人处理将相同的方法和途径应用于更大的样本队列来验证已发现的生物标记物(S)。如果多个蛋白质生物标记物得到验证，将开发用于糖尿病的AMS面板。这将通过构建多分析物亲和吸管来实现，该吸管将选择性地检索区分健康和疾病状态的目标生物标记物。这些小组还将接受与其每个组成部分所经历的相同的验证程序。这项研究的最终结果将是经过验证的2型糖尿病前期和2型糖尿病的蛋白质生物标记物，以及准备用于临床筛查和疾病诊断的技术。

DESCRIPTION (provided by applicant): The objective of this proposal is to apply novel affinity mass spectrometry (AMS) technologies to identify protein biomarkers for pre-diabetes or type 2 diabetes. The methodology used combines micro-affinity protein capture from complex biological solutions with mass spectrometric detection. In this first phase (R21), we will screen the provided plasma and blood cell samples to identify candidate proteins, and protein profiles, that can appropriately differentiate sample origin from 1 of the 3 groups (normal, pre-diabetic and diabetic; as determined by the administration of an oral glucose tolerance test). Both plasma and blood cell samples will be assayed with affinity pipettes derivatized with poly- and monoclonal antibodies for specific protein target analysis. Additionally, groups of proteins will be analyzed via hydrophilic and hydrophobic ligands, metal chelating ligands, and other wide-specificity affinity surfaces. Each of these ligand surfaces possesses specific affinity characteristics that will result in the capture and purification of individual or whole groups of proteins. Protein variations observed as a result of qualitative and/or quantitative differences, that have statistical significance and correctly group samples according to their origin, will be viewed as potential biomarkers. In the second (validation) phase (R33), we will validate the discovered biomarker(s) via the application of the same methods and approaches to a larger group of sample cohorts through the use of high throughput parallel robotic processing. In the event that multiple protein biomarkers are validated, AMS panels for diabetes will be developed. This will be achieved through the construction of multi-analyte affinity pipettes that will selectively retrieve the targeted biomarkers that differentiate healthy from diseased states. Such panels will also be subject to the same validation process that each of its components endured. The ultimate result of this research will be validated protein biomarkers for pre-and type 2 diabetes and technology that are ready for clinical screening and diagnosis of the disease.