Sensitive and Quantitative MS-bases Glycomic Mapping Platform

基于 MS 的灵敏定量糖组图谱平台

• 批准号: 10019565
• 负责人: Yehia Mechref
• 金额: $ 28.4万
• 依托单位: TEXAS TECH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10019565
• 关键词: Acids; Adhesions; Algorithms; Amino Acids; Area; Asparagine; Automation; Biological; Biological Assay; Biological Process; Biology; Carbon; Carbon nanoparticle; Cell Culture Techniques; Cell Differentiation process; Cells; Chromatography; Computer software; Cultured Cells; Cystic Fibrosis; Data; Data Set; Degenerative polyarthritis; Detection; Deuterium; Development; Diagnostic; Digestion; Disease; Drug Targeting; Energy Transfer; Enzymes; Evaluation; Exhibits; Funding; Future; Glycoproteins; Glycosides; Goals; Human; Immune response; Immune system; Isomerism; Isotope Labeling; Isotopes; Label; Laboratories; Light; Link; Lipids; Liquid Chromatography; Liquid substance; Malignant Neoplasms; Malignant neoplasm of esophagus; Malignant neoplasm of liver; Manuals; Mass Spectrum Analysis; Medical; Metabolic; Methods; Modeling; Monitor; Monosaccharides; Names; Natural graphite; Peptide N-glycohydrolase F; Phase; Physiological; Plasma; Polysaccharides; Preparation; Proteins; Proteomics; Public Health; Reaction; Recombinant Proteins; Reporting; Research; Research Personnel; Running; Sampling; Scientist; Serine; Signal Transduction; Signaling Protein; Site; Software Tools; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; System; Threonine; Tissue Sample; Tissues; Tyrosine; Variant; alpha-Fetoproteins; ammonium hydroxide; analytical method; analytical tool; base; cancer biomarkers; carbohydrate structure; experimental study; glycoproteomics; glycosylation; graphene; improved; informatics tool; interest; ionization; isotope incorporation; malignant breast neoplasm; methyl iodide; microwave electromagnetic radiation; open source; pathogen; preservation; rapid technique; side effect; software development; stable isotope; therapeutic protein; tool; tool development

Glycomics has emerged as an interesting yet challenging area of research in biology. Glycans function in numerous important biological areas such as, but not limited to: the immune system, cell development, cell differentiation/adhesion, host-pathogen interactions, protein signaling, and protein stabilization. Abnormal glycosylation has been associated with several diseases including cancer, cystic fibrosis, and osteoarthritis. Glycomics/glycoproteomics studies aim to quantify and characterize glycan structures (including linkage and positional isomers), protein attachment sites, and the protein’s identity. Approximately 50% of mammalian proteins are glycosylated but their abundance is rather low compared to non-glycosylated proteins. Furthermore, numerous glycans can occupy the same glycan attachment site on a protein; that is the same protein pool can have several different types of glycans attached to the same site, each with a potentially different function or a particular activity. Protein glycans are divided into two classes based on their amino acid attachment sites: asparagine for N-glycans and threonine, serine, and tyrosine for O-glycans. A strategy that has been successfully employed to investigate N-glycans in cells is to release or separate the glycans from proteins with the enzyme PNGase F, and study the global glycan composition of a sample. A drawback to this approach is that, so called, native glycans possess low ionization efficiencies which make their analysis by mass spectrometry quite difficult; however, this sensitivity issue can be overcome by permethylating glycans. Glycans have many isomers which can make their accurate analysis by LC-MS/MS difficult if the isomers cannot be resolved. This proposal demonstrates that we are able to separate permethylated glycan isomerss with a heated PGC column before mass spectrometry analysis (Aim 1), resulting in an extremely sensitive assay to accurately characterize and quantitate glycan isomers in biological samples. Although the separation of isomeric glycans has been previously reported, prior studies only resolved native and reducing end labeled glycan structures. Owing to the fact that permethylated glycans exhibit ionization efficiencies at least two orders of magnitude higher than the aforementioned structures, the importance of the increase in sensitivity, for the detection of structures at physiological concentrations, that accompanies isomeric separation of permethylated glycans (Aim 1) cannot be overstated. To overcome the variation in ionization efficiency between LC-MS samples, we have successfully permethylated glycans with various stable isotope combinations to achieve unprecedented quantitative glycan comparisons across samples derived from cell culture experiments, biological fluids, and biological tissues. Through the implementation of our multi-level isotopic labeling strategies (metabolic 15N labeling, 18O reducing end labeling and multiplex permethylation), the number of potential multiplexed samples can be increased from eight, the previous maximum, to 16 and 32 for biological fluid and tissue samples and cell culture samples, respectively (Aim 2). High throughput isomeric characterization glycans derived from biological samples can be attained by combining the methods described in Aims 1 and 2. While PNGase F is used extensively to release N-glycans from proteins, no such enzyme exists or has been discovered for O-glycans. We have developed a rapid method, RAIDR (Rapid Ammonium hydroxide Isobutyric acid O-glycan Deglycosylation Reaction), for selectively releasing O-glycans; RAIDR leaves the protein and N-glycans unscathed which allows for compatible downstream analyses (Aim 3). In addition to improving LC-MS analytical methods, we are also proposing the addition of graphene nanosheets and carbon nanoparticles to MALDI matrices for enhanced sample preparation, cleanup, and an increase in the ionization efficiencies of both native and permethylated glycans (Aim 4). Mass spectrometry based experiments can generate a tremendous amount of data that is cumbersome to analyze manually. There are numerous well-known proteomic software packages available but few that can comprehensively analyze glycomic datasets. We have developed MultiGlycan to analyze glycomic datasets and intend to expand its functionalities (Aim 5) to handle glycan isomers (Aim 1), multiplexed permethylated glycans (Aim 2), O-glycans (Aim 3), and glycans analyzed with MALDI-MS (Aim 4). The development of the proposed methods and algorithms will help us and collaborators to better understand the attributes and biomedical significance of glycan isomers in the development and progression of esophagus, breast, and liver cancer. We are also expecting the analytical tools and algorithms proposed here to be beneficial to other scientists who are interested in understanding the biological attributes of glycan isomers in other systems to benefit from.

糖基因成为生物学研究的一个有趣但挑战领域。糖的功能 许多重要的生物学领域，例如但不限于：免疫系统，细胞发育，细胞 分化/粘附，宿主 - 病原体相互作用，蛋白质信号传导和蛋白质稳定。异常 糖基化与多种疾病有关，包括癌症，囊性纤维化和骨关节炎。 糖/糖蛋白质组学研究旨在量化和表征聚糖结构（包括联系和 位置异构体），蛋白质附着位点和蛋白质的身份。哺乳动物约50％ 蛋白质是糖基化的，但与非糖基化蛋白相比，其丰度相当低。此外， 许多聚糖可以在蛋白质上占据相同的聚糖附着位点。那是相同的蛋白质池 在同一站点上有几种不同类型的糖，每个糖具有潜在的功能或 特定活动。蛋白质聚糖根据其氨基酸附着位点分为两类： 天冬酰胺用于N-聚糖和苏氨酸，丝氨酸和酪氨酸的O-聚糖。 成功地研究细胞中N-聚糖的策略是释放或分离 来自蛋白质的蛋白质的聚糖，并研究样品的全球聚糖组成。一个 这种方法的缺点是，所谓的本地聚糖具有低电离效率，这使他们 质谱分析非常困难；但是，可以通过氯甲基克服这种敏感性问题 聚糖。 Glycans具有许多异构体，如果很难通过LC-MS/MS进行准确的分析，如果 异构体无法解决。该提议表明我们能够分离苄苄氨基化聚糖 在质谱分析之前具有加热PGC柱的异构体（AIM 1），导致极度 敏感评估以准确表征和定量生物样品中的聚糖异构体。虽然 以前已经报道了异构聚糖的分离，先前的研究仅解决了天然和减少端 标记的聚糖结构。由于苄氨基化聚糖具有电离效率至少两个事实 比近似结构高的数量级，灵敏度增加的重要性， 为了在物理浓度下检测结构，涉及异构分离 苄苄化聚糖（AIM 1）不能被夸大。 为了克服LC-MS样品之间电离效率的变化，我们已经成功 具有各种稳定同位素组合的苄氯二甲基化聚糖，以实现前所未有的定量聚糖 跨细胞培养实验，生物液和生物组织的样品进行了比较。 通过实施我们的多级同位素标签策略（代谢15N标签，18O降低 最终标记和多重苄苄氨基化），可以增加潜在多路复用样品的数量 从八个（前面的最大值）到生物流体和组织样品以及细胞培养的16和32 样品分别（目标2）。高通量异构体特征源自生物学的聚糖 可以通过组合AIMS 1和2中描述的方法来附加样品。使用PNGase F 从蛋白质中释放出N-聚糖，没有这种酶存在或已被发现用于O-聚糖。 我们已经开发了一种快速方法RAIDR（快速氢氧化铵异丁酸O-聚糖 脱糖基化反应），用于选择性释放O-聚糖； RAIDR离开蛋白质和N-糖果 毫发无损，可以进行兼容的下游分析（AIM 3）。除了改善LC-MS分析 方法，我们还建议将石墨烯纳米片和碳纳米颗粒添加到Maldi 用于增强样品制备，清理和两者电离效率提高的矩阵 和苄苄化聚糖（AIM 4）。基于质谱的实验可以产生大量 手动分析的数据很麻烦。有许多著名的蛋白质组织软件包 可用，但很少能全面分析糖果数据集。我们已经开发了多聚糖 分析糖基胶质数据集并打算扩大其功能（AIM 5）来处理聚糖异构体（AIM 1）， 用MALDI-MS分析的多甲基化聚糖（AIM 2），O-Glycans（AIM 3）和聚糖（AIM 4）。 提出的方法和算法的开发将帮助我们和合作者更好地理解 聚糖异构体在食道和进展中的属性和生物医学意义， 乳腺癌和肝癌。我们还期望在这里提出的分析工具和算法是有益的 对于其他有兴趣了解其他系统中聚糖异构体的生物学属性的科学家 从中受益。