Novel Materials and Methods for Separations of Glycopeptides and Glycans

描述（由申请人提供）：糖蛋白多肽碳水化合物附着位点和这些蛋白质上存在的聚糖结构的分析需要高分辨率色谱分离和复杂的质谱分析相结合。亲水相互作用液相色谱法（HILIC）在糖肽和释放聚糖的分离中有很大的应用，对糖型的分离具有显著的选择性。提出的努力是显着提高分离糖肽和释放聚糖的HILIC，减少分离时间从目前的2-3小时每个样品，到30分钟或更少。糖蛋白LC/MS分析的分离组分可以受益于更高效的固定相色谱材料的设计，以及使用允许与质谱仪直接和高效接口的条件。我们提出，最近开发的表面多孔二氧化硅微颗粒二氧化硅包装材料（熔芯(R)结构）可以实现这些分离速度，而不会损失所需的高分辨率。AMT熔融核反相材料在小分子分离方面表现出优异的动力学性能和柱效率，最近，更宽孔径的填料在较大的肽（约3-5 kDa）中也表现出类似的优势。目前的提案使用具有独特表面修饰的fusion - core技术来开发高性能的HILIC材料，专门用于糖肽和聚糖的分离。量身定制的HILIC材料将被合成并测试用于HILIC操作，以及建立表面性质，颗粒特性和聚糖和糖肽分离效用之间的基本关系。

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.