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: 9814307
• 负责人: Peggi M Angel
• 金额: $ 28.17万
• 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/9814307
• 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 可用于快速有效地操纵数百个表达的技术 聚糖生物合成基因来研究复杂聚糖在生长细胞中的功能作用 使评估每个细胞表型的分析能力黯然失色 定量的方式。目前的糖组分析方法需要专门的基于板的 样品处理资源、分析前的广泛处理和纯化，以及 就处理时间和酶而言，成本昂贵。这些都是非糖组的障碍 和糖组学研究人员采用大规模聚糖分析工作流程应用于生物流体和 细胞。我们的合作小组最近开发了一种简化的抗体捕获载玻片阵列 直接分析捕获的血清糖蛋白（如 IgG）的 N-聚糖的方法，一种方法 需要几微升的样品和简化的处理工作流程，无需 分析前的纯化或糖修饰。 N-聚糖从捕获物中释放出来 糖蛋白并通过 MALDI-TOF 质谱直接分析。我们建议扩大 并调整我们基于载玻片阵列的免疫捕获工作流程，直接从 生物流体，并提供培养细胞的快速分析工作流程。这些测定的目标是 通过最少的处理和直接聚糖分析开发快速分离工作流程，例如 描述了三个具体目标：SA1。开发用于聚糖分析的载玻片方法 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以进行直接聚糖测量。优化 将强调载玻片化学和处理的原理，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程，例如 任何拥有 MALDI 质谱仪的研究或核心实验室都能够执行 对最常见类型的样品进行常规聚糖分析。