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

• 批准号: 8455999
• 负责人: Barry E Boyes
• 金额: $ 46.64万
• 依托单位: ADVANCED MATERIALS TECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8455999
• 关键词: Biological Process; Biology; Carbohydrates; Characteristics; Chemicals; Chemistry; Communicable Diseases; Complex; Complex Mixtures; Disease; Disease Marker; Exhibits; Experimental Designs; Glycopeptides; Glycoproteins; Goals; High Pressure Liquid Chromatography; Hour; Influenza; Kinetics; Knowledge; Liquid Chromatography; 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; Proteomics; Resolution; Sampling; Science; Series; Silicon Dioxide; Site; Speed; Structure; Surface; Surface Properties; Technology; Testing; Thick; Time; Work; analytical tool; cost; design; glycosylation; improved; liquid chromatography mass spectrometry; mass spectrometer; meetings; nanoparticle; new technology; novel; operation; particle; polypeptide; protein structure; public health relevance; real world application; research study; small molecule; sugar; trend

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.

描述（由申请人提供）：对碳水化合物附着的糖蛋白多肽位点和这些蛋白质上存在的聚糖结构的分析需要结合高分辨率色谱分离和复杂的质谱分析。亲水相互作用液相色谱 (HILIC) 在糖肽和释放聚糖的分离中具有很大的用途，对糖型的分离表现出显着的选择性。拟议的努力是显着改善 HILIC 对糖肽和释放聚糖的分离，将分离时间从目前每个样品 2-3 小时减少到 30 分钟或更短。糖蛋白 LC/MS 分析的分离组件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且高效连接的条件。我们建议，最近开发的表面多孔二氧化硅微粒二氧化硅填料（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 Fused-Core 反相材料此前已被证明在小分子分离方面表现出优异的动力学特性和柱效，最近，更宽孔径的填料对于较大的肽（约 3-5 kDa）也显示出类似的优势。目前的提案使用具有独特表面修饰的 Fused-Core 技术来开发专门用于分离糖肽和聚糖的高性能 HILIC 材料。定制的 HILIC 材料将被合成并测试用于 HILIC 操作，并建立表面性质、颗粒特征以及聚糖和糖肽分离效用之间的基本关系。 新的键合相利用了第一阶段研究中发现的有机硅烷化学物质，这些化学物质被选择来承受恶劣的使用条件，从而获得可在广泛条件下使用的稳定且坚固的分离材料。糖蛋白组学应用中的初步 LC/MS 应用已分阶段完成。合理的估计是，分离时间提高了 2 倍以上。第二阶段将扩大这一努力，旨在提供进一步改进的材料和方法，以满足糖蛋白质组学和蛋白质组学工作流程中更广泛的需求。 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。这项工作的目标是生产坚固的材料，允许与在线质谱分析简单集成，有效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。