Analytical tools for the analysis of clustered O-glycans in clinical samples

描述（由申请人提供）：糖基化是蛋白质最常见的翻译后修饰之一。据估计，超过一半的哺乳动物蛋白质是糖基化的。患有几种自身免疫性疾病、慢性炎性疾病和一些感染性疾病的患者表现出血清免疫球蛋白和其他糖蛋白的异常糖基化。这些修饰在健康和疾病中的生物学功能已成为生物医学研究中的重要领域。这些糖蛋白的一个子集具有在氨基酸序列内具有富含丝氨酸和苏氨酸的延伸的O-糖基化的聚集位点。粘蛋白，如膜相关MUC 1，可能是最著名的蛋白质家族，是严重的O-糖基化。它们在癌症中的改变的表达和异常的糖基化使它们成为早期检测疾病的生物标志物的潜在靶标。免疫球蛋白A1（IgA 1）含有O-和N-聚糖。IgA 1的异常O-糖基化参与伊加肾病（IgAN）的发病机制。有趣的是，异常糖基化的分子，IgAN中的IgA 1和癌症中的MUC 1，被免疫系统识别为新表位，如特异性抗体的形成所证明的。由于成簇的丝氨酸、苏氨酸和通常的脯氨酸残基，定位和表征这类糖蛋白内的整个范围的0-聚糖附着位点在分析上具有挑战性。我们最近开发了使用高分辨率质谱和电子捕获（或转移）解离串联质谱评估簇状IgA 1 O-聚糖宏观异质性（30个氨基酸区域内连接的O-聚糖的范围和分布）和微观异质性（同一区域内每个氨基酸位点的O-聚糖链的范围和分布）的方案。我们最近在这一挑战方面的进展使我们认识到，如果具有O-糖基化簇集位点的蛋白质要成为可靠的生物标志物，则需要标准化用于分析临床样品中的O-聚糖簇集的一系列分析工具。我们提出了以下具体目标，为临床样品中这类后修饰蛋白提供标准化的指导：1）从以IgAN患者为中心的一系列临床样品中确定IgA 1蛋白中O-糖基化簇合位点的一级结构; 2）确定由五种不同凝集素识别的簇合O-聚糖结构的范围;和3）开发用于定量评估单个蛋白质和肽簇O-糖型的策略。

DESCRIPTION (provided by applicant): Glycosylation is one of the most common post-translational modifications of proteins. It is estimated that over half of mammalian proteins are glycosylated. Patients with several autoimmune disorders, chronic inflammatory diseases, and some infectious diseases exhibit abnormal glycosylation of serum immunoglobulins and other glycoproteins. The biological functions of these modifications in health and disease have become a significant area of interest in biomedical research. A subset of these glycoproteins has clustered sites of O-glycosylation with serine- and threonine-rich stretches within the amino acid sequence. Mucins, such as membrane-associated MUC1, are perhaps the best known family of proteins that are heavily O-glycosylated. Their altered expression and aberrant glycosylation in cancer have made them potential targets as biomarkers for early detection of the disease. Immunoglobulin A1 (IgA1) contains both O- and N- glycans. Aberrant O-glycosylation of IgA1 is involved in the pathogenesis of IgA nephropathy (IgAN). Interestingly, the aberrantly glycosylated molecules, IgA1 in IgAN and MUC1 in cancer, are recognized by the immune system as neoepitopes, as evidenced by formation of specific antibodies. Locating and characterizing the entire range of O-glycan attachment sites within this class of glycoproteins is analytically challenging due to the clustered serine, threonine, and often proline residues. We have recently developed protocols for the assessment of clustered IgA1 O-glycan macroheterogeneity (range and distribution of O-glycans attached within a 30-amino acid region) and microheterogeneity (range and distribution of O-glycan chains at each amino acid site within the same region) by use of high-resolution mass spectrometry and electron capture (or transfer) dissociation tandem mass spectrometry. Our recent progress with this challenge has led to the realization that a series of analytical tools for the analysis of clustered O-glycans in clinical samples needs to be standardized if proteins with clustered sites of O-glycosylation are to become reliable biomarkers. We propose the following specific aims to provide standardized guidelines for this class of post-translationally modified proteins in clinical samples: 1) Define the primary structure of clustered sites of O-glycosylation in IgA1 proteins from a series of clinical samples centered around patients with IgAN; 2) Define the range of clustered O-glycan structures that are recognized by five different lectins; and 3) Develop strategies for the quantitative assessment of individual protein and peptide clustered O-glycoforms.