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）：糖基化是最常见的蛋白质翻译后修饰之一。据估计，超过一半的哺乳动物蛋白质被糖基化。患有多种自身免疫性疾病、慢性炎症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。这些改变对健康和疾病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聚集的 O-糖基化位点，其氨基酸序列内富含丝氨酸和苏氨酸。粘蛋白，例如膜相关 MUC1，可能是最知名的重度 O-糖基化蛋白家族。它们在癌症中的表达改变和异常糖基化使它们成为早期检测疾病的生物标志物的潜在目标。免疫球蛋白 A1 (IgA1) 含有 O- 和 N- 聚糖。 IgA1 的异常 O-糖基化参与 IgA 肾病 (IgAN) 的发病机制。有趣的是，异常糖基化分子，IgAN 中的 IgA1 和癌症中的 MUC1，被免疫系统识别为新表位，特异性抗体的形成证明了这一点。由于丝氨酸、苏氨酸和脯氨酸残基簇集，定位和表征此类糖蛋白中整个范围的 O-聚糖附着位点在分析上具有挑战性。我们最近开发了通过使用高分辨率质谱和电子捕获（或转移）解离串联质谱来评估聚集的 IgA1 O-聚糖宏观异质性（30 个氨基酸区域内附着的 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取得的进展使我们认识到，如果具有 O-糖基化聚集位点的蛋白质要成为可靠的生物标志物，则需要对临床样品中聚集的 O-聚糖分析的一系列分析工具进行标准化。我们提出以下具体目标，为临床样本中的此类翻译后修饰蛋白提供标准化指南：1）定义来自以 IgAN 患者为中心的一系列临床样本的 IgA1 蛋白中 O-糖基化聚集位点的一级结构； 2) 定义五种不同凝集素识别的簇状O-聚糖结构的范围； 3) 制定定量评估单个蛋白质和肽簇 O-糖型的策略。