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

基本信息

批准号：
9980821
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
项目类别：
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/9980821
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

项目摘要

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。