Mass Spectrometry Tools in Pursuit of Salivary Biomarkers of Sjogren's Syndrome

描述（由申请人提供）：我们提出使用基于质谱（MS）的方法的组合来发现和验证全唾液中原发性干燥综合征（pSS）的候选生物标志物。我们专注于pSS，因为临床上非常需要一种明确的非侵入性检测方法来诊断这种使人衰弱的疾病。因此，我们建议测试的假设，pSS外分泌蛋白病是伴随着唾液蛋白质组和/或糖组的改变，MS可以用来确定这些疾病相关的变化。我们将使用从Sj gren国际合作临床联盟（SICCA）获得的全唾液样本。在最近完成的实验中，我们比较了从Sj gren患者和对照个体收集的唾液样本的组成。在蛋白质水平上，我们发现了几种组分相对丰度的疾病相关变化。此外，免疫印迹法揭示了PSS相关的糖基化变化。具体而言，在pSS患者的样本中，我们检测到一致高水平的不寻常的碳水化合物结构，即在N-乙酰葡糖胺的6位上具有硫酸化作用的唾液酸刘易斯x，其在血液白细胞募集期间介导滚动和束缚。这些发现表明唾液成分中存在与疾病相关的变化，可以用作生物标志物。拟议的工作包括生物标志物发现过程的发现和验证阶段，第二阶段是开发临床免疫测定和验证标志物组，即，超出了计划的实验范围。具体而言，在目标1，发现阶段，我们将采用非靶向和靶向MS为基础的策略，以确定pSS的生物标志物。在非靶向方法中，由pSS患者和对照个体的全唾液样品产生的肽混合物将用同量异位素质量标签标记（iTRAQ试剂）和MS调查的深层蛋白质组。在靶向方法中，我们将在糖肽水平上使用凝集素和抗体亲和层析，并使用基于MS的测序来发现pSS-1。唾液蛋白糖基化的相关差异，并沿肽骨架定位修饰位点沿着。在目标2（验证阶段）中，我们将在可能的情况下使用免疫测定法，或者在没有免疫测定法的情况下，使用多反应监测（MRM）MS来定量来自其他pSS患者和对照受试者的更大组全唾液样本中的蛋白质，这些样本也是由SICCA收集的。因此，在所提出的实验结束时，我们将鉴定出pSS的候选生物标志物，其可以导致用于诊断这种病症的非侵入性临床测试。此外，这些实验的结果可能会产生其他重要的结果。例如，我们发现的生物标志物的子集也可能有助于诊断患有其他自身免疫性疾病的患者的SS。最后，疾病相关的变化，唾液蛋白的组成和/或糖基化可以给我们有趣的机制的见解，这种鲜为人知的条件的发病机制。

DESCRIPTION (provided by applicant): We propose using a combination of mass spectrometry (MS)-based approaches to discover and verify candidate biomarkers of primary Sj¿gren's Syndrome (pSS) in whole saliva. We are focusing on pSS due to a very large clinical need for a definitive non-invasive assay for diagnosing this debilitating disease. Accordingly, we propose testing the hypothesis that pSS exocrinopathy is accompanied by alterations in the salivary proteome and/or glycome and that MS can be used to identify these disease-related changes. We will utilize whole saliva samples obtained from the Sj¿gren's International Collaborative Clinical Alliance (SICCA). In recently completed experiments, we compared the composition of saliva samples collected from Sj¿gren's patients and control individuals. At the protein level, we discovered disease-related alterations in the relative abundances of several components. Additionally, an immunoblotting approach revealed pSS-associated changes in glycosylation. Specifically, in samples from pSS patients, we detected uniformly high levels of the unusual carbohydrate structure, sialyl Lewis x with sulfation on the 6-position of N-acetylglucosamine, which mediates rolling and tethering during leukocyte recruitment from the blood. These findings suggested the existence of disease-related changes in salivary composition that can be exploited as biomarkers. The proposed work includes the discovery and verification phases of the biomarker discovery process with development of a clinical immunoassay and validation of a marker panel as the second stage, i.e., outside the scope of the planned experiments. Specifically, in Aim 1, the discovery phase, we will employ untargeted and targeted MS-based strategies to identify biomarkers of pSS. In the untargeted approach, peptide mixtures that are generated from whole saliva samples of pSS patients and control individuals will be labeled with isobaric mass tags (iTRAQ reagents) and the deep proteome surveyed by MS. In targeted approaches, we will use lectin and antibody affinity chromatography at the glycopeptide level with MS-based sequencing to discover pSS-associated differences in the glycosylation of salivary proteins and to locate the modified sites along the peptide backbone. In Aim 2, the verification phase, we will use immunoassays when possible or, in their absence, multiple-reaction monitoring (MRM) MS to quantify proteins in a larger set of whole saliva samples, also collected by SICCA, from additional pSS patients and control subjects. Thus, at the conclusion of the proposed experiments we will have identified candidate biomarkers of pSS that could lead to a noninvasive clinical test for diagnosing this condition. In addition, the results of these experiments could have other important outcomes. For example, a subset of the biomarkers we discover may also be useful for diagnosing SS in patients who have other autoimmune diseases. Finally, disease-related changes in the composition and/or glycosylation of salivary proteins could give us interesting mechanistic insights into the pathogenesis of this poorly understood condition.