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（MS）的方法组合来发现和验证全唾液中原发性格林综合征（pSS）的候选生物标志物。我们之所以专注于pSS，是因为临床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，我们建议验证pSS外源性疾病伴随着唾液蛋白质组和/或糖的改变的假设，并且MS可以用于识别这些疾病相关的变化。我们将利用从Sj¿gren的国际合作临床联盟（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，我们发现了几种成分的相对丰度与疾病相关的变化。此外，免疫印迹方法显示pss相关的糖基化变化。具体来说，在来自pSS患者的样本中，我们检测到一致高水平的异常碳水化合物结构，在n -乙酰氨基葡萄糖的6位上磺化的sialyl Lewis x，它在白细胞从血液中募集过程中介导滚动和系缚。这些发现表明存在与疾病相关的唾液成分变化，可以作为生物标志物加以利用。拟议的工作包括生物标志物发现过程的发现和验证阶段，其中临床免疫测定的开发和标记面板的验证作为第二阶段，即在计划的实验范围之外。具体来说，在Aim 1的发现阶段，我们将采用非靶向和靶向MS-based策略来识别pSS的生物标志物。在非靶向方法中，从pSS患者和对照个体的整个唾液样本中产生的肽混合物将使用等压质量标签（iTRAQ试剂）和ms调查的深层蛋白质组进行标记。在靶向方法中，我们将使用凝集素和抗体亲和层析在糖肽水平上使用MS-based测序来发现pSS相关的唾液蛋白糖基化差异，并定位肽主链上的修饰位点。在Aim 2验证阶段，我们将在可能的情况下使用免疫测定，或者在没有免疫测定的情况下，使用多反应监测（MRM）质谱法来定量来自其他pSS患者和对照受试者的更大组完整唾液样本中的蛋白质，这些样本也由SICCA收集。因此，在实验的最后，我们将确定pSS的候选生物标志物，这可能导致诊断这种疾病的无创临床试验。此外，这些实验的结果可能有其他重要的结果。例如，我们发现的一个生物标志物子集也可能对患有其他自身免疫性疾病的患者的SS诊断有用。最后，唾液蛋白组成和/或糖基化的疾病相关变化可以为我们提供有趣的机制见解，以了解这种鲜为人知的疾病的发病机制。

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.