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

用于分析临床样品中聚集的 O-聚糖的分析工具

• 批准号: 8537478
• 负责人: Matthew B Renfrow
• 金额: $ 27.83万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8537478
• 关键词: Affect; Affinity; Amino Acid Sequence; Amino Acids; Antibodies; Antigen-Antibody Complex; Area; Autoimmune Diseases; Binding; Biological Assay; Biological Markers; Biological Process; Biomedical Research; Chronic; Clinical; Cluster Analysis; Communicable Diseases; Disease; Disease Marker; Dissociation; Early Diagnosis; Electrons; Exhibits; Glycopeptides; Glycoproteins; Goals; Health; IgA1; Immune system; Immunoglobulin A; Immunoglobulins; Individual; Inflammatory; Kidney Diseases; Lectin; Link; Lupus Nephritis; Malignant Neoplasms; Masks; Mass Spectrum Analysis; Measurement; Membrane; Modification; Mucin-1 Staining Method; Mucins; Myeloma Proteins; Normal Statistical Distribution; Parotid Gland; Pathogenesis; Patients; Peptides; Polysaccharides; Post-Translational Protein Processing; Precipitation; Proline; Protein Analysis; Protein Family; Protein Isoforms; Proteins; Protocols documentation; Recruitment Activity; Resolution; Saliva; Salivary; Sampling; Series; Serine; Serum; Sialic Acids; Site; Stretching; Structure; Surface Plasmon Resonance; Threonine; Time; activator 1 protein; analytical tool; assay development; base; disorder control; glycosylation; interest; jacalin; multiple reaction monitoring; protein distribution; sample collection; standardize guidelines; tandem mass spectrometry; volunteer

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.

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是糖基化的。患有多种自身免疫性疾病，慢性炎性疾病和一些传染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的丝氨酸和雌激素富含丝氨酸和苏氨酸伸展的O-糖基化的位点。粘蛋白，例如与膜相关的MUC1，也许是最著名的O型O-糖基化的蛋白质家族。它们在癌症中的表达改变和异常的糖基化使它们成为早期发现该疾病的生物标志物的潜在靶标。免疫球蛋白A1（IGA1）均包含O-和N-糖。 IgA1的异常O-糖基化参与IgA肾病（Igan）的发病机理。有趣的是，免疫系统将异常的糖基化分子，IgAN中的IgA1和癌症中的MUC1识别为新表皮，这通过特定抗体的形成证明。由于聚集的丝氨酸，苏氨酸和通常的脯氨酸残基，在该类别的糖蛋白中定位和表征O-Glycan附着位点的整个范围在分析上具有挑战性。我们最近开发了用于评估簇的IgA1 O-Glycan宏观分子性（在30个氨基酸区域内附着的O-聚糖的范围和分布）和微分近期（通过在同一区域内每个氨基酸位点的O-Glycan链的范围和分布）来评估的方案。我们最近在这项挑战中取得的进展使人们意识到，如果蛋白质具有O-糖基化的聚类位点，则需要将一系列用于分析临床样品中的O-聚糖分析的分析工具，以成为可靠的生物标志物。我们提出以下具体旨在为临床样品中的这类翻译后修饰的蛋白提供标准化指南：1）定义来自IgA患者围绕Igan患者的一系列临床样本的IgA1蛋白中O-糖基化簇位点的主要结构； 2）定义由五种不同凝集素识别的聚类O-聚糖结构的范围； 3）制定策略来定量评估单个蛋白质和肽聚集的O-糖型。