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.

糖组学已成为生物学中一个有趣但具有挑战性的研究领域。 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性异常。 糖基化与多种疾病有关，包括癌症、囊性纤维化和骨关节炎。 糖组学/糖蛋白质组学研究旨在量化和表征聚糖结构（包括连接和 位置异构体）、蛋白质附着位点以及大约 50% 的哺乳动物蛋白质特性。 蛋白质被糖基化，但与非糖基化蛋白质相比，其丰度相当低。 许多聚糖可以占据蛋白质上的相同聚糖附着位点，即相同的蛋白质库可以； 有几种不同类型的聚糖附着在同一位点上，每种聚糖都具有潜在的不同功能或功能 蛋白质聚糖根据其氨基酸附着位点分为两类： N-聚糖为天冬酰胺，O-聚糖为苏氨酸、丝氨酸和酪氨酸。 已成功用于研究细胞中 N-聚糖的策略是释放或分离 使用 PNGase F 酶从蛋白质中提取聚糖，并研究样品 A 的整体聚糖组成。 这种方法的阻碍在于，所谓的天然聚糖具有低电离效率，这使得它们 质谱分析相当困难；然而，这个灵敏度问题可以通过全甲基化来克服。 聚糖有许多异构体，如果存在异构体，则很难通过 LC-MS/MS 对其进行准确分析。 该提案表明我们能够分离全甲基化聚糖。 在质谱分析（目标 1）之前使用加热的 PGC 柱分离异构体，从而产生极其 准确表征和定量生物样品中聚糖异构体的灵敏测定。 异构聚糖的分离先前已有报道，先前的研究仅解决了天然和还原末端 由于全甲基化聚糖具有至少两倍的电离效率。 比上述结构高出几个数量级，提高灵敏度的重要性， 用于检测生理浓度下的结构，伴随异构体分离 全甲基化聚糖（目标 1）怎么强调都不为过。 为了克服 LC-MS 样品之间电离效率的差异，我们成功地 全甲基化聚糖与各种稳定同位素组合实现前所未有的定量聚糖 比较来自细胞培养实验、生物液体和生物组织的样品。 通过实施我们的多级同位素标记策略（代谢 15N 标记、 18O 还原 末端标记和多重全甲基化），可以增加潜在多重样品的数量 对于生物液体和组织样本以及细胞培养，从之前的最大值 8 个增加到 16 个和 32 个 分别对来自生物的聚糖进行高通量异构表征（目标 2）。 可以通过结合目标1和目标2中描述的方法来获得样品。虽然使用PNGase F 一般来说，从蛋白质中释放 N-聚糖，不存在或尚未发现用于 O-聚糖的此类酶。 我们开发了一种快速方法，RAIDR（快速氢氧化铵异丁酸O-聚糖 RAIDR 留下蛋白质和 N-聚糖 完好无损，除了改进 LC-MS 分析之外，还可以进行兼容的下游分析（目标 3）。 方法，我们还建议在 MALDI 中添加石墨烯纳米片和碳纳米颗粒 用于增强样品制备、净化并提高两种天然物质的电离效率的基质 和全甲基化聚糖（目标 4）。 手动分析起来很麻烦的数据有许多著名的蛋白质组学软件包。 可用但很少可以全面分析糖组数据集，我们开发了 MultiGlycan 来进行分析。 分析糖组数据集并打算扩展其功能（目标 5）以处理聚糖异构体（目标 1）， 多重全甲基化聚糖（目标 2）、O-聚糖（目标 3）和使用 MALDI-MS 分析的聚糖（目标 4）。 所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 聚糖异构体在食管发育和进展中的属性和生物医学意义， 我们还期望这里提出的分析工具和算法是有益的。 给其他有兴趣了解其他系统中聚糖异构体的生物属性的科学家 从中受益。