Novel Materials and Methods for Separations of Glycopeptides and Glycans

描述（由申请方提供）：糖蛋白多肽的碳水化合物连接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（HILIC）在糖肽和糖链的分离中有着广泛的应用，对糖型的分离具有显著的选择性。提出的努力是显著改善通过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。糖蛋白的LC/MS分析的分离组分可以受益于更有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粒二氧化硅填充材料（Fused-Core（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融合核材料先前已经显示出对于小分子的分离表现出上级动力学性质和柱效率，并且最近，更宽孔径的填料已经显示出对于较大肽的类似益处（c. 3-5 kDa）。目前的提案使用具有独特表面改性的融合核技术来开发专门用于糖肽和聚糖分离的高性能HILIC材料。孔径和表面定制的HILIC材料将被合成和测试HILIC操作，以及建立表面性能，颗粒特性，和聚糖和糖肽分离的实用程序之间的基本关系。新的键合相将利用选择的有机硅烷试剂，以承受恶劣的使用条件（低pH值和高温），允许稳定和强大的分离材料，可用于广泛的条件。一个合理的估计是，与目前的方法相比，可以获得2-5倍的分离时间的改进。 目前的提案将通过在糖蛋白结构分析中的使用，进一步评估这些新分离材料的益处和实际应用。这项工作的目标是生产耐用的材料，允许简单的集成与在线质谱分析，有效的复杂的混合物，是典型的当前糖蛋白的识别和结构表征程序的决议。 公共卫生相关性：通过添加糖（称为糖基化）对蛋白质进行修饰在自然界中非常常见，并且这种修饰对蛋白质结构和生物功能表现出强烈的影响。在某些疾病中，最显著的是在癌症中，蛋白质的糖基化以不完全理解的方式改变，但是其足够不同以允许这种修饰用于识别疾病，并且在某些情况下对患者进行分层。目前的建议是利用材料科学和化学的新知识，以更快、更有效的方式发现更多、更高质量的糖基化疾病标志物。

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.