Simplified Glycan Profiling Workflows of Captured Immune Glycoproteins and Cells

捕获的免疫糖蛋白和细胞的简化聚糖分析工作流程

• 批准号: 10227699
• 负责人: Peggi M Angel
• 金额: $ 25.42万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10227699
• 关键词: Acute Disease; Address; Adopted; Antibodies; Archives; B-Lymphocytes; Biological Assay; Biological Response Modifiers; Biotechnology; Blood; Blood Platelets; CD Antigens; Cell Line; Cells; Chemistry; Chronic Disease; Cirrhosis; Complex; Cultured Cells; Data; Development; Diagnostic; Digestive System Disorders; Endothelial Cells; Enzymes; Epithelial Cells; Europe; Ficoll; Flow Cytometry; Genes; Genetic; Glass; Glycoproteins; Goals; Heart Diseases; IgG1; IgG2; IgG3; IgG4; Image; Immune; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Immunoglobulins; Isotope Labeling; Laboratories; Leukocytes; Link; MALDI-TOF Mass Spectrometry; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Metabolic; Methodology; Methods; Modification; Molecular; Peptide N-glycohydrolase F; Phenotype; Plasma; Polysaccharides; Reagent; Recombinants; Reporting; Research; Research Personnel; Resources; Rheumatoid Arthritis; Role; Sampling; Serum; Slide; Sorting - Cell Movement; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; T-Lymphocyte; Technology; Time; Tissues; Work; analytical method; base; cell type; cohort; comparative; glycosylation; improved; instrumentation; mass spectrometer; monocyte; sugar; tool

Abstract The technologies available to rapidly and efficiently manipulate the expression of hundreds of glycan biosynthetic genes to study the functional role of complex glycans in growing cells have eclipsed the analytical capabilities to evaluate each cell phenotype in a comparative or quantitative manner. Current glycome profiling approaches require specialized plate-based sample handling resources, extensive processing and purification prior to analysis, and are expensive in regards to processing time and enzyme. These are barriers to both non-glycomic and glycomic researchers adopting large scale glycan analysis workflows applied to biofluids and cells. Our collaborative group has recently developed a streamlined antibody capture slide array approach to directly profile N-glycans of captured serum glycoproteins like IgG, a method requiring a few microliters of sample and simplified processing workflows that require no purification or sugar modifications prior to analysis. N-glycans are released from captured glycoproteins and directly analyzed by MALDI-TOF mass spectrometry. We propose to expand and adapt our slide array-based immune capture workflows to isolate immune cells directly from biofluids, and provide rapid analysis workflows of cultured cells. The goal in these assays is to develop rapid isolation workflows with minimal processing and direct glycan analysis, as described in three Specific Aims: SA1. Development of an on slide method for glycan analysis of immunoglobulin subtypes: SA 2. Development of Glyco-Cell Typer as applied to immune cell sub-types. SA 3. Analysis of cultured cells on slides for direct glycan measurements. Optimization of slide chemistry and processing will be emphasized, as well as conditions for metabolic isotope labeling and quantitative glycan analysis. The goal will be to provide validated workflows such that any research or core laboratory with a MALDI mass spectrometer will be able to perform routine glycan analysis on the most common types of samples.

抽象的 可以快速有效地操纵数百种的表达的技术 聚糖生物合成基因研究复杂聚糖在生长细胞中的功能作用 在比较或 定量方式。当前的聚糖分析方法需要专门的基于板的专业 在分析之前进行样本处理资源，广泛的处理和净化，并且是 关于处理时间和酶的昂贵。这些都是非散热障碍的障碍 以及采用大规模聚糖分析的糖研究人员，应用于生物流体和 细胞。我们的协作小组最近开发了一个简化的抗体捕获幻灯片阵列 直接介绍捕获的血清糖蛋白（如IgG）的N-聚糖的方法，一种方法 需要几个微量的样本和简化的处理工作流，不需要 分析前进行纯化或糖修饰。 n-glycans从捕获中释放 糖蛋白并通过MALDI-TOF质谱法直接分析。我们建议扩展 并调整我们的基于幻灯片阵列的免疫捕获工作流程以直接分离免疫细胞 生物流体，并提供对培养细胞的快速分析工作流程。这些分析的目标是 通过最少的处理和直接的聚糖分析来开发快速隔离工作流程，作为 在三个特定目标中描述：SA1。开发用于聚糖分析的幻灯片方法 免疫球蛋白亚型：SA 2。应用于免疫细胞的Glyco-Cell Typer的开发 子类型。 SA 3。分析载玻片上的培养细胞进行直接聚糖测量。优化 将强调幻灯片化学和加工以及代谢同位素的条件 标记和定量聚糖分析。目标是提供经过验证的工作流 任何使用MALDI质谱仪的研究或核心实验室都将能够执行 对最常见的样品类型的常规聚糖分析。

项目成果

On the Cutting Edge of Translational Research.

论转化研究的前沿。

• DOI: 10.1093/clinchem/hvaa273
• 发表时间: 2021
• 期刊: Clinical chemistry
• 影响因子: 9.3
• 作者: Angel,PeggiM

GlycoFibroTyper: A Novel Method for the Glycan Analysis of IgG and the Development of a Biomarker Signature of Liver Fibrosis.

• DOI: 10.3389/fimmu.2022.797460
• 发表时间: 2022
• 期刊: Frontiers in immunology
• 影响因子: 7.3
• 作者: Scott DA;Wang M;Grauzam S;Pippin S;Black A;Angel PM;Drake RR;Castellino S;Kono Y;Rockey DC;Mehta AS
• 通讯作者: Mehta AS