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Sensitive and Quantitative MS-bases Glycomic Mapping Platform

基于 MS 的灵敏定量糖组图谱平台

基本信息

批准号：
10697345
负责人：
Yehia Mechref
金额：
$ 30.29万
依托单位：
TEXAS TECH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-15 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10697345
关键词：
2019-nCoV Address Adhesions Algae Algorithms Alzheimer&apos s Disease Anabolism Area Bacterial Infections Bioinformatics Biological Biological Process Blood capillaries COVID-19 Carbon Cell Differentiation process Cells Cirrhosis Clinical Code Communicable Diseases Communities Complex Computer software Coronavirus Coronavirus spike protein Coupled Cystic Fibrosis Development Diabetes Mellitus Disease Drug Targeting Fingerprint Future Gangliosides Genes Glucose Glycoconjugates Glycolipids Glycoproteins Goals Heterogeneity High temperature of physical object Human Genome Immune response Inflammation Investigation Ions Isomerism Isotopes Kidney Diseases Label Libraries Malignant Neoplasms Malignant neoplasm of esophagus Malignant neoplasm of liver Maps Mass Spectrum Analysis Metabolic Diseases Metastatic malignant neoplasm to brain Methods Monitor Nature Oligosaccharides Outcome Parkinson Disease Pattern Polysaccharides Porosity Post-Translational Protein Processing Process Protein Glycosylation Proteins Public Health Reaction Recombinant Proteins Research Research Activity Research Personnel Resolution Role Running Sampling Scientist Software Tools Symbiosis System Techniques Time Traumatic Brain Injury Variant Viral Virion Virus Diseases analytical tool base bioinformatics tool cancer biomarkers carbohydrate structure clinical diagnostics computerized data processing drug development glycosylation implementation tool improved indexing interest ionization machine learning prediction malignant breast neoplasm nano pathogen predictive signature protein complex side effect software development tandem mass spectrometry therapeutic protein tool tool development

项目摘要

Glycosylation is one of the most complex protein modifications; more than 50% of mammalian proteins are glycosylated. The fact that there are 100,000 proteoforms coded by only ~20,300 genes identified in the human genome emphasizes the importance of posttranslational modifications (PTMs) like glycosylation. Aberrant protein glycosylation has been implicated in many diseases, such as Alzheimer’s disease, congenital/metabolic disorders, diabetes, inflammation, Parkinson’s disease, bacterial/viral infectious diseases, and various cancers. More recently, glycans have been associated with coronavirus spike glycoproteins, including the SARS- CoV-2 virion. The diverse biological roles of glycans and their implications in diseases have created a demand for reliable qualitative and quantitative glycomic approaches, which facilitates sensitive investigation of glycan changes in different biological and biomedical samples. Mass spectrometry (MS) is the most efficient technique in glycomics due to its high sensitivity and capacity for acquiring structural information. However, glycomic research remains a challenge because of the microheterogeneity of glycan compositions in complex biological samples; the relatively low abundance in nature and low ionization efficiency in MS analysis; and the existence of variant positional and linkage isomers caused by the biosynthesis process. To overcome these challenges, several separation methods have been coupled to MS. Despite the development of these separation techniques, isomeric separation of glycans remains insufficient. There is an increasing demand for more efficient isomeric separation approaches since glycan isomers have been related to different diseases. The main aim of this proposal is to provide easily accessible, adaptable, and affordable strategies for better separation and characterization of glycans and glycan isomers derived from different glycoconjugates. Aim 1 is focused on finding a replacement for porous craphitic carbon columns that sufferes from low reprodcubility and loss of resolution and efficiency with time. The in-house mesoporous graphitic carbon (MGC)-LC-MS will be investigated for both permethylated (Aim 1a) and native (Aim 2b) isomeric separation of N- and O-glycans, glycolipid glycans, and free oligosaccharides. Other alternatives (also part of Aim 1a), such as 50 cm and 200 cm micro pillar array columns (μPAC)-C18-LC-MS, and a 50 cm long capillary nanoC18-LC-MS, will also be evaluated to achieve an improved isomeric separation of glycan isomers. Subsequently, GUI will be utilized to improve the identification of glycan isomers. A GUI libraries for the separation strategies developed in Aim 1 will be established to normalize the possible retention time shift among different runs (Aim 2). LC-M based glycomics quantitative strategies will be developed and assessed in Aim 3. The combination of permethylation and TMT will capitalize on the advantages of both techniques, providing enhanced sensitivity and accuracy in glycan quantitation (Aim 3a). Additionally, the combination of 8-plex isotopic permethylation and 6-plex TMT will facilitate a reliable 48-plex, thus significantly increasing the throughput of analysis (Aim 3b). Furthermore, with the improved separation and identification (Aim 1 and 2), PRM will be employed to assist the better quantitation of glycans and the differentiation of glycan isomers. A library containing the fragments fingerprint of each glycan isomer will be created to aid the glycan isomeric identification (Aim 3c). The development of automated software for easy, fast, and accurate glycomic data processing is the main focus of Aim 4. Finally, the aforementioned strategies and tools will be applied to clinical samples to address a variety of biological and biomedical issues such as COVID-19, breast cancer brain metastasis, liver cancer vs. cirrhosis, kidney diseases, algae development, and others (Aim 5). The development of the proposed methods and algorithms will help us and our collaborators to better understand the attributes and biomedical significance of glycan isomers in the development and progression of esophageal, breast, and liver cancer. Furthermore, we expect the analytical tools and algorithms proposed here to benefit researchers and scientists who are interested in understanding the biological attributes of glycan isomers in other systems.

糖基化是最复杂的蛋白质修饰之一；超过50%的哺乳动物蛋白质被糖基化。事实上，有 100,000 个蛋白质形式仅由约 20,300 个编码人类基因组中鉴定的基因强调了翻译后的重要性修饰 (PTM)，如糖基化。异常的蛋白质糖基化与许多疾病，例如阿尔茨海默病、先天性/代谢性疾病、糖尿病、炎症、帕金森病、细菌/病毒感染性疾病和各种癌症。更多的最近，聚糖与冠状病毒刺突糖蛋白有关，包括 SARS- CoV-2 病毒体。聚糖的多种生物学作用及其对疾病的影响产生了对可靠的定性和定量糖组方法的需求，这有助于对不同生物和生物医学样品中聚糖变化的敏感研究。大量的光谱测定法 (MS) 因其高灵敏度和高可靠性而成为糖组学中最有效的技术。获取结构信息的能力。然而，糖组学研究仍然是一个挑战由于复杂生物样品中聚糖成分的微观异质性；这自然界中的丰度相对较低，质谱分析中的电离效率较低；和存在由生物合成过程引起的位置异构体和连接异构体的变异。为了克服这些为了应对挑战，几种分离方法已与 MS 结合使用。尽管发展这些分离技术，聚糖的异构体分离仍然不够。有一个由于聚糖异构体已经存在，对更有效的异构体分离方法的需求不断增加与不同的疾病有关。该提案的主要目的是提供易于获取、适应性强且负担得起的更好地分离和表征聚糖和衍生自聚糖异构体的策略不同的糖复合物。目标 1 专注于寻找多孔石墨碳的替代品色谱柱的重现性较低，并且随着时间的推移，分辨率和效率会降低。这将研究内部介孔石墨碳 (MGC)-LC-MS 的全甲基化 N- 和 O- 聚糖、糖脂聚糖和天然 (目标 2b) 异构体分离游离寡糖。其他替代方案（也是目标 1a 的一部分），例如 50 厘米和 200 厘米微型柱阵列柱 (μPAC)-C18-LC-MS 和 50 cm 长的毛细管 nanoC18-LC-MS，还将进行评估以实现聚糖异构体的改进的异构体分离。随后，图形用户界面将用于改进聚糖异构体的鉴定。用于分离的 GUI 库将制定目标 1 中制定的策略，以使可能的保留时间变化正常化在不同的运行之间（目标 2）。将开发基于 LC-M 的糖组学定量策略并在目标 3 中进行评估。全甲基化和 TMT 的结合将利用两种技术的优点，提高了聚糖的灵敏度和准确性定量（目标 3a）。此外，8 重同位素全甲基化和 6 重同位素全甲基化的组合 TMT 将促进可靠的 48 重分析，从而显着提高分析吞吐量（目标 3b).此外，随着分离和识别的改进（目标 1 和 2），PRM 将用于帮助更好地定量聚糖和区分聚糖异构体。一个将创建包含每个聚糖异构体的片段指纹的文库以帮助聚糖异构体鉴定（目标 3c）。自动化软件的开发，方便、快速、准确的糖组数据处理是目标 4 的主要焦点。最后，前面提到的策略和工具将应用于临床样本，以解决各种生物和生物医学问题，例如 COVID-19、乳腺癌脑转移、肝癌与肝硬化、肾脏疾病、藻类发育等（目标 5）。拟议的发展方法和算法将帮助我们和我们的合作者更好地理解属性和聚糖异构体在食管发育和进展中的生物医学意义，乳腺癌、肝癌。此外，我们期望提出的分析工具和算法在这里让有兴趣了解生物学的研究人员和科学家受益其他系统中聚糖异构体的属性。

项目成果

期刊论文数量（85）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Implication of the Kallikrein-Kinin system in neurological disorders: Quest for potential biomarkers and mechanisms.

DOI：
10.1016/j.pneurobio.2018.01.003
发表时间：
2018-06
期刊：
Progress in neurobiology
影响因子：
6.7
作者：
Nokkari A;Abou-El-Hassan H;Mechref Y;Mondello S;Kindy MS;Jaffa AA;Kobeissy F
通讯作者：
Kobeissy F

LC-MS/MS Isomeric Profiling of N-Glycans Derived from Low-Abundant Serum Glycoproteins in Mild Cognitive Impairment Patients.

DOI：
10.3390/biom12111657
发表时间：
2022-11-08
期刊：
Biomolecules
影响因子：
5.5
作者：
通讯作者：

Heat Stress of Algal Partner Hinders Colonization Success and Alters the Algal Cell Surface Glycome in a Cnidarian-Algal Symbiosis.

DOI：
10.1128/spectrum.01567-22
发表时间：
2022-06-29
期刊：
MICROBIOLOGY SPECTRUM
影响因子：
3.7
作者：
Maruyama, Shumpei;Mandelare-Ruiz, Paige E.;McCauley, Mark;Peng, Wenjing;Cho, Byeong Gwan;Wang, Junyao;Mechref, Yehia;Loesgen, Sandra;Weis, Virginia M.
通讯作者：
Weis, Virginia M.

Isomeric separation of permethylated glycans by extra-long reversed-phase liquid chromatography (RPLC)-MS/MS.