氟试剂修饰N-糖组质谱精准分析新方法

N-glycomics is an important field in cancer early diagnoses, disease process monitor, prognosis evaluation and therapeutic target identification. Mass spectrometry (MS) has proven to be the most prominent methodology in analysis of N-glycome. However, due to its inherent low abundance and poor ionization efficiency, caused by its high hydrophilicity, N-glycan produces low signal in MS analysis. Besides, N-glycan is a non-template synthesis during its bio-synthesis, which results in a huge amount of N-glycan structures, making interpretation of its MS spectra a hard task. Though many enrichment/purification methods have been proposed by researchers, the enriching recovery, analysis sensitivity and structure identification ability of glycans by MS still remain to be improved. Based on our prior work, this project will propose a novel strategy for improved MS-analysis of N-glycan based on fluorous reagent derivatization of the reducing end of N-glycan. The introduction of a high-hydrophobicity fluorous reagent will increase the ionization efficiency of N-glycan by more than one order of magnitude and provide more kinds of fragment ions during MS/MS, which would assist in elucidating composition and structure of N-glycan. After reacting with the fluorous reagent, the N-glycan could be enriched via fluorous solid-state extraction. In summary, this novel strategy is a combination of N-glycan derivatization and enrichment, which aims to achieve higher sensitivity and structure identification accuracy in MS analysis of N-glycan. This project will focus on how reagent structure, reaction type, fluorous solid-state material and MS/MS fragment mode influence the MS analysis of derivatized N-glycan. A new method would be established based on experiment results and applied to N-glycome analysis of serum from human colorectal cancer patient.

N-糖组研究已经成为肿瘤的早期诊断、进程监测、预后评估以及治疗靶点寻找的重要领域。质谱技术是N-糖组研究的重要手段，然而，由于糖链丰度低、亲水性强在质谱中离子化效率低；糖链非模板合成，组成和结构复杂，质谱数据解析难度大。现有糖链富集纯化方法的选择性、回收率，质谱分析灵敏度和结构解析能力都亟待进一步提高。项目拟在前期基础上，提出采用氟试剂衍生糖链还原端醛基并质谱分析的新方法。高疏水性氟试剂的引入将使糖链在质谱中的离子化效率显著提高1个数量级以上，并在串级质谱中提供丰富的碎片离子，辅助糖链的组成和结构解析；氟试剂衍生后可通过氟-氟相互作用对糖链进行高选择性的富集纯化。因此新方法将一步实现N-糖组的衍生、富集、离子化和结构解析的精准分析。项目重点研究修饰分子类型、反应基团、和含氟固相富集材料及在不同碎裂模式下对分析效率的影响，总结形成规律并将建立的方法应用于人大肠癌血清N-糖组研究。

N-糖链是除核酸、蛋白质类的另外一类重要的生物大分子，在许多生命过程包括细胞-细胞、细胞-基质的相互作用，蛋白质折叠，和受体结合中发挥重要的作用。发展和建立糖链高精准质谱分析方法及相关技术是糖链得以准确定性定量研究的重要前提。本项目发展了一系列包括含氟同位素分子标记N-糖链富集鉴定和相对定量的新方法，双端稳定同位素标记同时定性定量唾液酸和中性N-糖链的新方法以及金属伪同位素标记的N-糖链高通量定性定量方法等。以上方法在糖链质谱定性分析方面提高了酸性糖链的稳定性和离子化效率，并基于稳定同位素提供的分子量差异实现了唾液酸个数的确认；在定量方面同位素标记完全，在两个数量级线性范围内具有良好的定量准确性和重现性(R2>0.99, CV<20%)。所发展的方法成功用于分析大肠癌等病人血清N-糖链的变化，发现在肠癌病人血清样品中N-糖链分支变多且核心岩藻糖化、唾液酸化水平增加。