酶化学策略解析O-连接糖蛋白质的质谱新方法研究

Protein glycosylation is one of the most common protein modifications. Compared with N-linked glycoprotein, O-linked glycans are more diverse, glycosylation site has no consensus sequence, and no enzyme exists that can cleavage all kinds of O-glycans from their attached sites. These features make O-glycoprotein analysis one of the most challenging projects. In this proposal, on the basis of our newly developed NGAG method, we intend to develop a new OGAG method that can be used for comprehensive analysis of O-linked glycoproteins. As all peptides were first immobilized onto aldehyde-beads in the NGAG method, and the N-linked glycans and glycosite-containing peptides were then released sequentially from the beads for N-glycosylation analysis. In this study, we will further release O-linked glycans and glycosite-containing peptides from the beads using β-elimination method and Lys-N/Lys-C digestion, respectively. A modified approach will also be developed to enrich TMT-labeled intact O-glycopeptides, and our GPQuest software will be updated and optimized for TMT-label quantification. This new method will be applied in the analysis of protein expression and O-glycosylation alterations when the cells were under stress caused by O-glycosylation inhibition. This new method combined with our previous NGAG method will achieve simultaneous analysis of N- and O-linked glycans, glycosite-containing peptides, glycoproteins, and glycans attached to specific glycosites via intact glycopeptides, which would be a powerful tool for the structure and function analysis of glycoproteins as well as the biomarker discovery and disease-related mechanism studies.

蛋白糖基化是最常见的蛋白质修饰之一。与N-糖蛋白相比，O-糖蛋白糖链结构更加多样、糖基化位点没有保守序列、且没有可切除全部O-糖链的O-糖肽酶。这些特征使得O-糖蛋白质分析成为最具挑战性的课题之一。本项目中，我们将在前期NGAG方法学的基础上进一步建立可用于全面分析O-糖蛋白质组的OGAG新方法。NGAG方法首先将样品中所有的多肽固定到醛基化树脂上，然后依次释放N-糖链和去糖基多肽。本项目中我们将继续使用β-消除-加成反应和Lys-N/Lys-C酶切依次释放O-糖链和去糖基化多肽，并完成对TMT标记O-糖肽的富集、质谱分析方法的优化和完整糖肽分析软件GPQuest的优化升级。新方法将用于细胞对O-蛋白糖基化抑制的应激调控研究。OGAG联合NGAG方法将可实现对同一样本中N-和O-糖链、糖蛋白、糖基化位点和完整糖肽的全面分析，为糖蛋白结构和功能分析、新的肿瘤标志物发现和疾病发病机理研究提供强有力的糖蛋白质组学分析方法。

蛋白糖基化是最常见的蛋白质修饰之一。与N-糖蛋白相比，O-糖蛋白糖链结构更加多样、糖基化位点没有保守序列、且没有可切除全部O-糖链的O-糖肽酶。这些特征使得O-糖蛋白质难以分析，亟需可实现对O-连接糖蛋白全面系统分析的方法。为此，本项目中我们建立了基于二甲胺β-消除、氨基乙硫醇加成和Trypsin酶切的OGAG方法，实现对同一样本的O-糖、糖基化位点及去糖基化多肽全面系统分析，可为糖蛋白结构和功能分析、新的肿瘤标志物发现和疾病发病机理研究提供强有力的支持。将OGAG方法应用到人血清和人唾液样本中，以1%FDR 作为阈值，18种糖链、581条去糖基化多肽被鉴定到，其中311条多肽由O-糖基化位点修饰成后鉴定而来。随后，我们对O-糖肽的质谱分析方法进行优化，使得在一次质谱结果中，完整O-糖肽包括糖链、多肽以及糖基化位点得信息得以鉴定。并对完整糖肽分析软件GPQuest进行升级，增加了基于OGAG 方法鉴定到的O-糖链和去糖基化多肽数据库，实现对完整O-糖肽的识别和鉴定。人血清和人唾液样本共有209个完整O-糖肽被鉴定到（1%FDR ）。最后，我们将整套方法应用于O-蛋白糖基化抑制后的HT-29细胞，对其进行定性和定量分析，根据时序变化，对O-糖基化抑制反应的显著改变的蛋白质进行生物学功能及相关通路分析，阐述了O-糖基化抑制后蛋白的改变与可能的分子机制。这些数据为之后O-糖基化抑制后细胞的应激调控机制提供了宝贵的数据资源。本项目发表SCI 论文10篇，培养博士研究生3 人，硕士研究生8人。