微量样品蛋白质组糖基化高灵敏度分析新技术新方法

Glycoproteome analysis is crucial to the investigation of various biological processes, such as immune response and cell communication, as well as the diagnosis and treatment of serious diseases, such as cancer. However, current technologies and methods for glycoproteome analysis usually need more than milligrams of protein samples and there is still lack of reliable strategy for highly sensitive analysis of microgram and sub-microgram levels protein samples. We have developed many new technologies and methods for minute protein samle.proteolysis, glycopeptides enrichment and deglycosylation, and online multidimensinoal lquid chromatography separation in our previous works. We propose to develop new technologies and methods for highly sensitive glycoproteome analysis in this project, including new methods for high efficient protein extraction from minute amount of biological samples, new materials for high specific glycoproteins or glycopeptides enrichment, development of integrated glycoproteome analysis micro-reactor, and high resolution pre-fractionation and.online multidimensional liquid chromatography coupled with tandem mass spectrometry analysis. We will feasibly improve the sensitivity for highly sensitive glycoproteome analysis through this project, and develop reliable technologies for microgram and sub-microgram levels protein sample analyses, which will provide solid technology support for the glycoproteome analysis of minute amount of biological and clinical samples.

蛋白质组糖基化分析对于研究免疫应答、细胞通讯等生物学过程和癌症等重大疾病的诊断、治疗意义重大。 现有的蛋白质组糖基化分析技术和方法基本上还是针对大量的毫克级蛋白质样品，对微克和亚微克级的蛋白质样品还没有成熟稳定的糖基化组学分析技术，极大限制了其对重要微量生物学样品的分析。申请人在前期的研究工作中发展了多种针对微量蛋白质样品的酶解、糖基化多肽富集及去糖基化、在线多维色谱分离等新技术新方法。本项目针对微量蛋白质样品发展蛋白质组糖基化高灵敏度分析新技术新方法，拟从微量蛋白质样品高效提取，糖基化蛋白质和多肽高效、高选择性富集，构建糖基化分析微反应器，以及微量糖基化蛋白质样品高效预分级和高分辨色谱分离四个方面开展。通过本项目的开展，将有效提高蛋白质组糖基化分析的灵敏度，在国际上率先实现微克和亚微克级蛋白质样品的高效糖基化组学分析，为生物学和临床医学中微量生物样品蛋白质组糖基化分析提供技术支持。

本项目的研究内容为蛋白质组糖基化高灵敏度分析检测新技术新方法，项目目标为实现蛋白质组糖基化的高灵敏度分析。项目研究团队从糖基化多肽高效富集分离、高灵敏度质谱检测、高效二级质谱解离、高准确定定量分析等多个方面围绕项目目标开展研究工作；项目的主要研究成果包括建立了水溶性金团簇均相富集-共沉淀反应体系，消除了糖基化多肽富集过程中的传质阻力，实现了均相富集材料和糖基化多肽的共沉淀，糖基化多肽检测灵敏度达到50 amol；发展了基于密闭式离子源的高浓度有机气氛辅助电喷雾离子化新方法，实现了酶解多肽质谱检测信号平均提高5倍；发展了金团簇辅助激光解吸附离子化新方法，多肽、寡糖、脂类等分子检测质谱信号平均提高36倍；发展了紫外激光解离方法，实现了多肽骨架高效碎裂而不发生翻译后修饰的中性丢失，实现了序列和修饰位点的准确分析；发展了多肽和整体蛋白质稳定同位素标记定量新方法，显著提高了蛋白质组学定量分析的通量和准确性；发展了活性蛋白质赖氨酸标记策略，实现了高通量定量分析微克级膜受体、激酶等靶蛋白与小分子配体或抑制剂结合时不同蛋白区域的构象变化程度，揭示了小分子-靶蛋白的互作界面和蛋白构象调节模式。项目发表通讯作者SCI论文18篇，包括Chemical Science 1篇，Analytical Chemistry 5篇，ACS Nano 1篇，Trends in Analytical Chemistry 1篇，Journal of Physical Chemistry Letters 1篇，Chemical Communications 2篇；申请中国发明专利13件，培养博士研究生3名，相关工作完成了项目预定目标。