Multidimensional chromatographic separation and mass spectrometry of glycoprotein

描述(申请人提供)：糖蛋白等蛋白质的翻译后修饰变体，其中许多对蛋白质治疗和疾病诊断很重要，由于其丰度和结构分布较低，对传统的2D凝胶和毛细管液相色谱构成了严峻的挑战。开发一种快速、准确的技术来表征这些蛋白质是当务之急。我们的第一阶段结果证明了利用自组装纳米颗粒或胶体晶体作为超高分辨率色谱介质的可行性，以及在2D微流控设备中执行多烷色谱分离的可能性。我们建议进一步努力，系统地将胶体晶体集成到可商业化的、功能齐全的设备中，并使用多烷样品注射器，并展示使用纳米光谱和基质辅助激光解吸和电离(MALDI)质谱仪分离和检测糖蛋白及其胰酶消化液的改进水平。这些拟议努力的结果，如果成功，将成为从消耗品到系统的原型产品，这些产品将极大地改进不仅对糖蛋白感兴趣的从业者，而且还对整个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性能和速度都出众的全新的基于设备的商业产品。蛋白质的糖基化程度在控制信号和细胞间识别方面具有广泛的重要性。糖蛋白是疾病的生物标记物，包括许多癌症。对于血红蛋白来说，糖基化的程度与糖尿病或长期应激有关。在像促红细胞生成素这样的药物中，一种用于治疗各种贫血和其他疾病的数十亿美元的糖蛋白产品仍然是一个重大挑战，如果给患者的剂量中的糖基化不正确，他们可能会死亡。分析蛋白质糖基化水平的能力是缺乏的，这将使对这一重要过程的理解和临床分析中生物标记物的检测取得进展。这项提议旨在创建有效和快速的工具来进行这种分析。

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.