Simplified Glycan Profiling Workflows of Captured Immune Glycoproteins and Cells

摘要 现有的技术可以快速有效地操纵数百种基因的表达， 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， 掩盖了分析能力，以评估每个细胞表型在一个比较或 量化的方式。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， 在加工时间和酶方面昂贵。这些都是非糖代谢的障碍， 和糖组学研究人员采用应用于生物流体的大规模聚糖分析工作流程， 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 直接分析捕获的血清糖蛋白如IgG的N-聚糖的方法， 需要几微升的样品和简化的处理工作流程， 在分析之前进行纯化或糖修饰。N-聚糖从捕获的 糖蛋白并通过MALDI-TOF质谱法直接分析。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， 生物流体，并提供培养细胞的快速分析工作流程。这些试验的目的是 开发快速分离工作流程，只需最少的处理和直接聚糖分析， 在三个具体目标中描述：SA 1。玻片法测定葡萄糖含量的研究 免疫球蛋白亚型：SA 2。糖细胞分型仪在免疫细胞中的应用 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，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， 任何拥有MALDI质谱仪的研究或核心实验室都能够进行 对最常见类型的样本进行常规聚糖分析。

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.