Novel Materials and Methods for Separations of Glycopeptides and Glycans

描述(由申请人提供)：分析糖结合的糖蛋白多肽位置和这些蛋白质上存在的葡聚糖结构需要结合高分辨率色谱分离和复杂的质谱学。亲水作用液相色谱(HILIC)在分离糖肽和释放的多糖方面有很大的应用，对糖类化合物的分离表现出显著的选择性。拟议的努力是显著改进HILIC对糖肽和释放的多糖的分离，将每个样品的分离时间从目前的2-3小时减少到30分钟或更短。糖蛋白的LC/MS分析的分离部分可以得益于更高效的固定相色谱材料的设计，以及允许直接和高效地与质谱仪对接的条件的使用。我们建议，最近开发的表面多孔二氧化硅微粒二氧化硅填充材料(熔芯(R)结构)可以达到这些分离速度，而不会损失所需的高分辨率。AMT熔核反相材料此前已被证明在分离小分子方面表现出优越的动力学性能和柱效，最近，更宽的孔径填料对较大的多肽(约3-5 kDa)也显示出类似的好处。目前的方案使用融合核心技术和独特的表面修饰来开发专门用于糖肽和糖聚糖分离的高性能HILIC材料。将合成定制的HILIC材料，并针对HILIC操作进行测试，以及建立表面特性、颗粒特性和用于分离多糖和糖肽的基本关系。 新的键合相利用了在第一阶段工作中发现的有机硅烷化学，这些有机硅烷已经被选择来承受侵蚀性的使用条件，允许稳定和坚固的分离材料，这些材料可以在广泛的条件下使用。LC/MS在糖蛋白组学应用中的初步应用已分阶段完成。一个合理的估计是，分离时间提高了2倍。第二阶段将扩大这一努力，目的是向糖蛋白组学和蛋白质组学工作流程中更广泛的需求提供进一步改进的材料和方法。当前提案的一个重要方面将评估这些新分离材料的好处和实际应用情况。这项工作的目标是生产坚固的材料，允许简单地与在线质谱分析相结合，有效地拆分当前糖蛋白鉴定和结构表征程序中典型的复杂混合物。

DESCRIPTION (provided by applicant): Analysis of glycoprotein polypeptide sites of carbohydrate attachment and the structures of the glycans present on these proteins requires the combination of high resolution chromatographic separations and sophisticated mass spectrometry. Hydrophilic interaction liquid chromatography (HILIC) has great use in separations of glycopeptides and released glycans, exhibiting remarkable selectivity for the separations of glycoforms. The proposed effort is to markedly improve separations of glycopeptides and released glycans by HILIC, reducing the separation times from the current 2-3 hours per sample, to 30 minutes or less. The separations component of LC/MS analysis of glycoproteins can benefit from the design of more efficient stationary phase chromatographic materials, and from the use of conditions that allow direct and productive interfacing with mass spectrometers. We propose that recently developed superficially-porous silica microparticulate silica packing materials (Fused-Core(R) structures) can achieve these separation speeds, without loss of the required high resolution. The AMT Fused-Core reversed phase materials have previously been shown to exhibit superior kinetic properties and column efficiencies for separations of small molecules, and recently, wider pore size packings have shown a similar benefit for larger peptides (c. 3-5 kDa). The current proposal uses Fused-Core technology with unique surface modifications to develop high performance HILIC materials specifically for separations of glycopeptides and glycans. Tailored HILIC materials will be synthesized and tested for HILIC operation, as well as to establish the fundamental relationships between surface properties, particle characteristics, and utility for glycan and glycopeptides separations. The new bonded phases utilize organosilane chemistry discovered in Phase I efforts that have been selected to withstand aggressive conditions of use, permitting stable and robust separations materials that can be used across a broad range of conditions. Preliminary LC/MS applications in glycoproteomic applications have been completed in Phase. A reasonable estimate is that an improvement of >2-fold for separations times are obtained. Phase II will expand on this effort, with the purpose of delivering further improved materials and methods to a broader range of needs in glycoproteomics and proteomics workflows. A significant aspect of the current proposal will assess the benefits and real-world applications of these new separations materials. The target of this work is to produce robust materials that permit simple integration with online mass spectrometry analysis, effective for resolution of the complex mixtures that are typical of current glycoprotein identification and structural characterization procedures.