Simplified Glycan Profiling Workflows of Captured Immune Glycoproteins and Cells

摘要 可以用来快速有效地处理数百个 研究多聚糖生物合成基因在生长细胞中的功能作用 在比较或比较中评估每种细胞表型的分析能力相形见绌 数量化的方式。目前的糖糖图谱分析方法需要专门的平板 样品处理资源，分析前的广泛处理和纯化，以及 在处理时间和酶方面都很昂贵。这些都是非血糖的障碍 和糖学研究人员采用大规模的糖链分析工作流应用于生物体液和 细胞。我们的合作小组最近开发了一种流线型抗体捕获载玻片阵列 一种直接提取免疫球蛋白等血清糖蛋白N-糖链的方法 需要几微升的样品和简化的处理工作流程，不需要 分析前的提纯或糖改性。从捕获的产品中释放N-葡聚糖 糖蛋白，直接用MALDI-TOF质谱仪分析。我们计划扩大 并调整我们基于幻灯片阵列的免疫捕获工作流，以直接从 生物流体，并提供培养细胞的快速分析工作流程。这些化验的目标是 开发快速分离工作流程，只需最少的处理和直接的多糖分析，如 描述了三个具体目标：SA1。琼脂糖凝胶分析方法的建立 免疫球蛋白亚型：Sa-2.应用于免疫细胞的Glyco-cell Typer的发展 子类型。SA 3.直接测定糖链的玻片上培养细胞的分析。优化 将强调载玻片的化学和加工，以及代谢同位素的条件 标记和定量多聚糖分析。我们的目标是提供经过验证的工作流，例如 任何拥有MALDI质谱计的研究或核心实验室都将能够执行 对最常见类型的样品进行常规的葡聚糖分析。

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.