Multidimensional chromatographic separation and mass spectrometry of glycoprotein

描述（由申请人提供）：蛋白质如糖蛋白的后修饰变体，其中许多对于蛋白质治疗和疾病诊断是重要的，由于其低丰度和结构分布，对常规2D凝胶和毛细管液相色谱提出了严峻的挑战。迫切需要开发一种快速准确的技术来表征这些蛋白质。我们的第一阶段结果已经证明了利用自组装纳米颗粒或胶体晶体作为高分辨率色谱介质的可行性，以及在2D微流体装置中进行多通道色谱分离的可能性。我们建议进一步这些努力，系统地实现集成的胶体晶体到商业化的，功能齐全的设备与多通道进样器，并证明在分离和检测的糖蛋白和胰蛋白酶的胰蛋白酶的水平的改善使用纳米喷雾和基质辅助激光解吸电离（MALDI）质谱。如果成功的话，这些拟议的努力的结果将是从消耗品到系统的原型产品，这些原型产品将大大改善不仅对糖蛋白感兴趣的从业者而且对蛋白质组学感兴趣的从业者可用的工具箱。该系列产品将帮助蛋白质组学从业者从目前的工作流程实践过渡到全新的基于设备的商业产品，这些产品具有上级性能和速度。蛋白质的糖基化程度在控制信号和细胞-细胞识别中广泛重要。糖蛋白作为疾病的生物标志物，包括许多癌症。对于血红蛋白，糖基化的程度与糖尿病或长期压力有关。在诸如红细胞生成素的药物中，用于治疗各种贫血和其他病症的数十亿美元的糖蛋白产品仍然是一个主要挑战，如果给予患者的剂量中的糖基化不正确，患者可能会死亡。缺乏分析蛋白质糖基化水平的能力，这将使对这一重要过程的理解和临床分析中生物标志物的检测取得进展。该提案旨在创建有效和快速进行这种分析的工具。

DESCRIPTION (provided by applicant): The post-translationally modified variants of proteins such as glycoproteins, many of which are important for protein therapeutics and disease diagnostics, pose severe challenges to conventional 2D gels and capillary liquid chromatography because of their low abundance and distribution of structures. Developing a fast and accurate technology to characterize these proteins is an urgent need. Our Phase 1 results have demonstrated the feasibility of utilizing self-assembled nanoparticles, or colloidal crystals, as an ultrahigh resolution chromatographic medium, and the possibility of performing multilane chromatographic separation in a 2D microfluidic device. We propose to further these efforts to systematically achieve integration of the colloidal crystals into commercializable, fully functional devices with multilane sample injectors, and to demonstrate the level of improvement in the separation and detection of glycoproteins and their tryptic digests using both nanospray and matrix-assisted laser desorption and ionization (MALDI) mass spectrometry. The results of these proposed efforts, if successful, will be prototype products from consumables to systems that dramatically improve the toolbox available not only to practitioners interested in glycoproteins, but also in proteomics in general. The series of products will help transition the proteomics practitioners from their current work flow practice to entirely new device-based commercial products of superior performance and speed. The extent of glycosylation of proteins is broadly important in controlling signal and cell-cell recognition. Glycoproteins serve as biomarkers for disease, including many cancers. For hemoglobin, the extent of glycosylation is correlated with diabetes or prolonged stress. In a drug such as erythropoetin, a multibillion-dollar glycoprotein product used for treatment of various anemias and other conditions remains a major challenge, in the patients may die if the glycosylation in the dose given to them is not correct. The ability to analyze glycosylation levels of proteins, which is lacking, would allow advances in the understanding of this important process and the detection of biomarkers in clinical analysis. This proposal aims to create tools that will be effective and high speed for this analysis.