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

描述(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。一些自身免疫性疾病、慢性炎症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糖基化异常。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的一个重要领域。这些糖蛋白的一个子集具有O-糖基化位点，在氨基酸序列中具有富含丝氨酸和苏氨酸的延伸部分。粘蛋白，如膜相关的MUC1，可能是最广为人知的高度O-糖基化的蛋白质家族。它们在癌症中的表达变化和异常糖基化使其成为早期发现疾病的潜在生物标记物。免疫球蛋白A1(IgA1)同时含有O-糖链和N-糖链。IgA1异常的O-糖基化参与了IgA肾病的发病过程。有趣的是，异常糖基化的分子，如IgAN中的IgA1和癌症中的MUC1，被免疫系统识别为新的表位，这是通过形成特定的抗体来证明的。由于成簇的丝氨酸、苏氨酸和经常是脯氨酸残基，在这类糖蛋白中定位和表征整个范围的O-葡聚糖连接位点在分析上是具有挑战性的。我们最近开发了使用高分辨率质谱仪和电子捕获(或转移)解离串联质谱仪来评估簇状IgA1 O-葡聚糖宏观异质性(连接在30个氨基酸区域内的O-葡聚糖的范围和分布)和微观异质性(同一区域内每个氨基酸位置的O-葡聚糖链的范围和分布)的方法。我们在这一挑战方面的最新进展使我们认识到，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，一系列用于分析临床样本中簇状O-糖链的分析工具需要标准化。我们提出了以下具体目标，以在临床样本中为这类翻译后修饰蛋白提供标准化指南：1)定义以IgAN患者为中心的一系列临床样本中IgA1蛋白O-糖基化的聚集位点的一级结构；2)定义由五种不同凝集素识别的聚集O-糖链结构的范围；以及3)开发单个蛋白质和多肽聚集O-糖形式的定量评估策略。

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.