Mass Defect-based Chemical Tags for Multiplex Glycan Quantitation

用于多重聚糖定量的基于质量缺陷的化学标签

• 批准号: 9352747
• 负责人: LINGJUN LI
• 金额: $ 29.49万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-15 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9352747
• 关键词: Address; Amination; Amines; Biological; Biological Process; Capillary Electrophoresis; Carbohydrates; Cardiovascular system; Cell Adhesion; Cells; Charge; Chemicals; Communities; Complex; Coupled; Custom; Data Set; Defect; Detection; Development; Digestion; Disease; Disease Pathway; Gases; Glycoproteins; Goals; Heterogeneity; Image; Immune System Diseases; Individual; Ions; Isotopes; LIF gene; Label; Lead; Ligand Binding; Link; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Methods; Neurodegenerative Disorders; Oligosaccharides; Optics; Ornithine; Pathway interactions; Peptide N-glycohydrolase F; Performance; Phase; Play; Polysaccharides; Post-Translational Protein Processing; Procedures; Protein Analysis; Protein Glycosylation; Proteins; Quinazolines; Reaction; Reagent; Research; Research Personnel; Resolution; Role; Sampling; Serum; Signal Transduction; Site; Source; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; System; Technology; Variant; base; clinically relevant; cost effective; experimental study; functional group; glycoproteomics; glycosylation; human disease; imaging platform; improved; innovation; instrumentation; intercellular communication; ion mobility; ionization; multiplex detection; novel; pancreatic cancer cells; pressure; protein transport; receptor; response; tool; two-dimensional

ABSTRACT Protein glycosylation plays critical roles in many biological processes as one of the most common and the most complex posttranslational modifications. Alterations in glycosylation profiles are known to be associated with many diseases, including cancer, neurodegenerative disorders, immunological diseases and cardiovascular problems. Advances in mass spectrometry (MS)-based glycoproteomics and glycomics are increasingly enabling qualitative and quantitative approaches for site-specific structural analysis of protein glycosylation. However, quantitative analysis of native glycans remains extremely challenging due to high complexity and heterogeneity and diversity of glycan structures, the low response in both optical and mass spectrometric detection methods, and the wide dynamic range of glycans in clinically relevant samples. Analytical strategies for N-linked glycans from glycoproteins usually involve releasing glycans from the glycoproteins via PNGase F digestion and labeling the released glycans with reagents that facilitate detection. The primary goal of this proposal is to develop a novel and cost effective chemical labeling technology via the custom synthesis and development of a set of mass defect-based tags that enable both sensitive fluorescent detection and high-throughput quantification of complex glycans with high resolution mass spectrometry (MS) platforms. We propose the following specific aims: Specific Aim 1 – To synthesize and develop novel mass defect-based multiplex dimethyl pyrimidinyl ornithine (DiPyrO) tags for cost effective and high-throughput MS- based relative quantification of N-glycans released from biological samples. Specific Aim 2 – To optimize the resolution of isomeric glycan structures and maximize the glycan ionization efficiency, nanoHILIC will be coupled with a novel sub-ambient pressure ionization nanospray (SPIN) source and high resolution Orbitrap MS platform for ultrasensitive detection of the multiplex DiPyrO labeled N-glycans released from complex biological samples such as serum and cell lysates. Specific Aim 3 – To synthesize and develop new mass defect-based multiplex dimethyl quinazolinyl ornithine (DiQuniO) tags for improved optical detection and develop capillary electrophoresis-UV/LIF-MALDI-ion mobility-mass spectrometric imaging platform for multi-dimensional glycan characterization and quantification. Collectively, our proposed experiments will develop new enabling tools and will generate cost-effective and novel mass defect-based labeling reagents for robust, sensitive and accurate glycan analysis with enhanced quantitative performance and structural elucidation capabilities.

摘要 蛋白质糖基化在许多生物过程中发挥着关键作用，它是最常见和最重要的 复杂的翻译后修饰。已知糖基化特征的改变与 许多疾病，包括癌症、神经退行性疾病、免疫性疾病和心血管疾病 有问题。基于MS的糖蛋白组学和糖组学研究进展日益增多 为蛋白质糖基化的特定部位结构分析提供定性和定量方法。 然而，由于高度的复杂性和复杂性，对天然多糖的定量分析仍然具有极大的挑战性。 多糖结构的异质性和多样性，在光学和质谱学中的低响应性 检测方法，以及临床相关样本中葡聚糖的广泛动态范围。分析策略 因为糖蛋白中的N-连接糖链通常涉及通过PNGase F从糖蛋白中释放出糖链 用便于检测的试剂消化和标记释放的多聚糖。这样做的主要目标是 建议通过定制合成开发一种新的、成本效益高的化学标记技术 以及开发一组基于质量缺陷的标签，使敏感的荧光检测 和高通量的高分辨质谱仪(MS)定量复杂多聚糖 站台。我们提出了以下具体目标：具体目标1-合成和开发新型质量 基于缺陷的多重二甲基嘧啶基鸟氨酸(DiPyrO)标签，用于经济高效和高通量的MS- 基于从生物样品中释放的N-糖链的相对定量。具体目标2--优化 拆分异构体的葡聚糖结构，最大限度地提高葡聚糖的电离效率，将纳米HILIC偶联 采用新型亚常压电离纳米(SPIN)源和高分辨率Orbitrap MS平台 用于超灵敏检测从复杂生物样品中释放的多重二吡咯标记的N-糖链 如血清和细胞裂解物。具体目标3-合成和开发基于质量缺陷的新型多路复用器 二甲基喹唑啉鸟氨酸(DiQuniO)标记用于改进光学检测和开发毛细管 电泳法-UV/LIF-MALDI-离子迁移率-多维多糖质谱联用成像平台 定性和量化。总体而言，我们提议的实验将开发新的使能工具和 将产生经济高效的基于质量缺陷的新型标记试剂，以实现健壮、灵敏和准确 具有增强的定量性能和结构阐明能力的葡聚糖分析。