Notch信号通路调控细胞的分化及凋亡等发育过程，其受体蛋白是Notch蛋白，存在N-和O-糖基化修饰，其中O-糖基化对Notch信号转导是必需的，O-糖链直接介导了Notch蛋白与其配体之间的相互结合。现有的研究主要集中于功能方面，对Notch蛋白O-糖组结构研究很少，主要是通过糖基转移酶水平等间接的推测分析得到的，缺乏结构生物学的直接的和更有力的证据，影响了对Notch蛋白与其配体蛋白相互作用的分子机制的研究。本项目利用本实验室已经建立的O-糖链非还原性解离和定量方法，以质谱为手段，全面解析果蝇Notch蛋白O-糖组的精细结构信息，包括O-糖组的种类、分子量分布、连接方式、序列等，系统分析不同发育阶段的表达量及糖型差异。本项目研究将进一步验证、补充和完善现有的对果蝇Notch蛋白O-糖组结构的推测，对进一步揭示Notch信号通路的分子机制有重要意义，也将为药物新靶标发现提供科学依据。

Notch signaling pathway regulates a variety of developmental processes including cell fate specification, differentiation, proliferation and apoptosis. Instead, derangement of the Notch signaling pathway play causative roles in a series of human disease, such as developmental disorders, aortic valve diseases, T-cell acute lymphoblastic leukemia, Alagille syndrome, heart disease and cancer. Notch proteins, receptors of the pathway, undergo N- and O-glycosylation, O-glycans of which mediate directly the binding between the Notch receptors and their ligands and are broadly considered to be essential for Notch signaling. The Notch protein N- glycans, however, haven't been found acting significantly in the Notch signaling process. Previous studies are mostly focused on the functions of O-glycans defined mainly by indirect presumption and analysis based on the level of glycosyltransferases, the structure of the Notch protein O-glycome remains to be investigated, lacking more direct and forcible structural biological proof and obstructing the research on the molecular mechanism of the interaction between Notch proteins and their ligands. Moreover, glycans are extremely hard to analyze using the traditional devices such as high-performance liquid chromatography (HPLC), gas chromatography (GC) and electrophoresis, due to their existence in very low abundance and with rather complex structure. However, mass spectrometry, especially biological mass spectrometry including electrospray ionization mass spectrometry (ESI-MS) and matrix assisted laser desorption and ionization mass spectrometry (MALDI-MS), provides a powerful tool for the structure analysis of glycans. This project, based on our previously established procedure of one-pot nonreductive O-glycan release and labeling and relative quantitation methods using stable isotopic labeling, aims to analyze comprehensively the detailed structural information of the Notch protein O-glycome including the O-glycan species, molecular weight distribution, linkage, sequence and glycosidic bond type, using Drosophila melanogaster as the model material and mass spectrometry as the detection device, and to compare systematically the differences of expression level, glycotype and detailed structure of the Notch protein O-glycome at diverse developmental stages and in various fruit fly cells, using the newly developed relative quantitation methods and high-performance liquid chromatography coupling with mass spectrometry (LC-MS) as the analytical instrument. This project is expected to validate, enrich and consummate further the present preliminary presumptions on the structure of the Notch protein O-glycome, to facilitate further investigations on structure-function correlations of the Notch protein O-glycome in flies, to play important roles in revealing the mechanism of the Notch signaling pathway, and to provide valuable reference for searching new targets for drugs development.