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。提出的努力是显着改善通过HILIC分离糖肽和释放的聚糖，将分离时间从目前的每个样品2-3小时减少到不到30分钟。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，更大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。孔径和表面定制的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。新的键合相将使用有机硅烷试剂，以承受恶劣的使用条件（低pH值和高温），允许稳定和坚固的分离材料，可在广泛的条件下使用。一个合理的估计是，与目前的方法相比，分离时间可以提高2-5倍。目前的提案将进一步评估这些新的分离材料在糖蛋白结构分析中的益处和实际应用。这项工作的目标是生产坚固的材料，允许简单地集成在线质谱分析，有效地解决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 less than 30 minutes. 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 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. Pore size and surface 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 will utilize organosilane reagents selected to withstand aggressive conditions of use (low pH and elevated temperatures), permitting stable and robust separations materials that can be used across a broad range of conditions. A reasonable estimate is that an improvement of 2-5 fold in separations times can be obtained, compared with current methods. The current proposal will further assess the benefits and real-world applications of these new separations materials by use in glycoprotein structural analysis. 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. PUBLIC HEALTH RELEVANCE: Protein modification by the addition of sugars (called glycosylation) is extremely common in nature, and this modification exhibits a strong effect on protein structure and on biological function. In certain diseases, most notably in cancer, glycosylation of proteins is altered in ways that are not fully understood, but which are sufficiently different to allow such modifications to be used for recognition of disease, and in some cases to stratify patients. The current proposal is to use new knowledge in materials science and chemistry to enable much faster and efficient ways to discover more, and higher quality glycosylation disease markers.