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Novel Materials and Methods for Separations of Glycopeptides and Glycans

用于分离糖肽和聚糖的新材料和方法

基本信息

批准号：
8124341
负责人：
Barry E Boyes
金额：
$ 19.96万
依托单位：
ADVANCED MATERIALS TECHNOLOGY, INC.
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-05-01 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8124341
关键词：
Biological Process Bos taurus structural-GP protein Carbohydrates Characteristics Chemicals Chemistry Complex Complex Mixtures Cost Analysis Disease Disease Marker Evaluation Exhibits Glycopeptides Glycoproteins Goals High Pressure Liquid Chromatography Hour Kinetics Knowledge Liquid Chromatography Liquid substance Malignant Neoplasms Marketing Mass Spectrum Analysis Measures Methods Modification Nature Outcome Patients Peptides Performance Phase Polysaccharides Post-Translational Protein Processing Procedures Property Protein Glycosylation Proteins Reagent Resolution Sampling Science Series Silicon Dioxide Site Speed Structure Surface Surface Properties Technology Temperature Testing Thick Time Work design glycosylation improved liquid chromatography mass spectrometry liquid chromatography mass spectroscopy mass spectrometer meetings nanoparticle new technology novel operation particle performance tests polypeptide protein structure prototype real world application research study small molecule sugar tool trend

项目摘要

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.

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分钟以下。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融合核心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。将合成孔径和表面量身定制的HILIC材料，并针对HILIC操作进行测试，以及建立表面特性、颗粒特征和用于分离多糖和糖肽的基本关系。新的键合相将利用经过挑选的有机硅烷试剂，以承受侵蚀性的使用条件(低pH值和高温)，从而允许稳定和坚固的分离材料，这些分离材料可以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，可以获得2-5倍的分离时间。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好处和现实应用。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。与公众健康相关：在自然界中，通过添加糖对蛋白质进行修饰(称为糖基化)是非常常见的，这种修饰对蛋白质结构和生物功能有很大的影响。在某些疾病中，尤其是在癌症中，蛋白质的糖基化以一种尚未完全了解的方式发生改变，但这种改变足够不同，使得这种改变可以用于识别疾病，在某些情况下还可以对患者进行分层。目前的建议是利用材料科学和化学方面的新知识，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。