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

描述(由申请人提供)：我们建议使用基于质谱仪(MS)的方法的组合来发现和验证全唾液中的原发性干燥综合征(PSS)的候选生物标记物。我们将重点放在PSS上，因为临床上需要一种明确的非侵入性检测来诊断这种令人衰弱的疾病。因此，我们建议检验这样一种假设，即PSS外分泌病伴随着唾液蛋白质组和/或糖类的改变，并且MS可以用于识别这些疾病相关的改变。我们将利用从圣何格伦国际合作临床联盟(SICCA)获得的全部唾液样本。在最近完成的实验中，我们比较了从Sjégren患者和对照组收集的唾液样本的组成。在蛋白质水平上，我们发现几种成分的相对丰度发生了与疾病相关的变化。此外，免疫印迹方法显示PSS相关的糖基化改变。具体地说，在PSS患者的样本中，我们检测到一致高水平的不同寻常的碳水化合物结构，唾液酸路易斯x与N-乙酰氨基葡萄糖6位上的硫酸盐化，它在白细胞从血液中重新募集时介导滚动和拴系。这些发现表明唾液成分中存在与疾病相关的变化，可用作生物标记物。拟议的工作包括生物标记物发现过程的发现和验证阶段，开发临床免疫分析和确认标记物小组是第二阶段，即不在计划的实验范围内。具体地说，在目标1，即发现阶段，我们将采用非靶向和靶向的MS策略来识别PSS的生物标记物。在非靶向方法中，从PSS患者和对照组的全唾液样本中产生的多肽混合物将被标记有等压质量标签(iTRAQ试剂)和MS调查的深层蛋白质组。在靶向方法中，我们将在糖肽水平使用凝集素和抗体亲和层析，并基于MS测序来发现PSS相关的唾液蛋白糖基化的差异，并沿着肽骨架定位修饰位点。在目标2，验证阶段，我们将在可能的情况下使用免疫分析，或者在没有免疫分析的情况下，使用多反应监测(MRM)MS来量化更大的一组完整唾液样本中的蛋白质，这些样本也是由SICCA从其他PSS患者和对照对象那里收集的。因此，在拟议的实验结束时，我们将确定PSS的候选生物标记物，这可能导致一种诊断这种情况的非侵入性临床测试。此外，这些实验的结果可能还有其他重要的结果。例如，我们发现的生物标记物的子集也可能对诊断患有其他自身免疫性疾病的患者的SS有用。最后，与疾病相关的唾液蛋白组成和/或糖基化的变化可能给我们提供关于这种鲜为人知的疾病的发病机制的有趣的机械性见解。

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.