氧化石墨烯固定化凝集素的制备及在糖蛋白/糖肽富集中的应用  

Preparation of graphene oxide based immobilized lectin and its application to efficient glycoprotein / glycopeptide enrichment

作　　者：时照梅 范超[2] 黄俊杰[2] 白海红[2] 秦伟捷[2] 蔡耘[1,2] 钱小红[2] 

机构地区：[1]安徽医科大学研究生学院,安徽合肥230032 [2]军事医学科学院放射与辐射医学研究所北京蛋白质组研究中心蛋白质组学国家重点实验室,北京102206

出　　处：《色谱》2015年第2期116-122,共7页Chinese Journal of Chromatography

基　　金：国家重大科学计划项目(2013CB911204);国家重大科学仪器设备开发专项项目(2011YQ09000504);国家自然科学基金项目(21275005;21235001)

摘　　要：生物体内蛋白质的糖基化修饰调控着细胞识别、细胞黏附和迁移以及免疫应答等多种生理过程,并与多种人类重大疾病的发生、发展密切相关。因此对蛋白质糖基化修饰的鉴定,不仅能够为生物学机理研究提供重要信息,对疾病诊断标志物和治疗靶标的发现也至关重要。然而在复杂生物体系中,大多数糖蛋白为低丰度蛋白质,其含量与现有质谱仪器的检测灵敏度之间存在较大差距,所以对含有不同糖型结构的糖蛋白进行全面/高效的富集,是实现高灵敏度糖蛋白鉴定的必由之路。凝集素富集作为一种有效的糖蛋白富集方法,已在糖蛋白质组学研究中得到了广泛的应用。针对现有凝集素功能化材料存在负载量偏低以及富集效率有限等问题,我们制备了两种以氧化石墨烯(GO)为载体的新型固定化凝集素,利用GO比表面积大,功能基团含量高,分散性、化学稳定性好等特点,实现了高负载量的凝集素固定(GO-ConA 2.073 mg/mg,RSD=1.0%;GO-WGA 1.908 mg/mg,RSD=0.14%)。同时考察了材料的可重复使用性与稳定性:每隔3天测一次同一GO-lectin材料对对应糖蛋白的富集效果,可以看出材料合成两周内富集效果都>200μg/mg。将该GO-lectin成功应用于糖蛋白、糖肽的选择性富集,在糖蛋白质组学研究中体现出良好的应用潜力。Protein glycosylation in eukaryotic cells regulates a variety of physiological proces- ses including cell recognition, cell adhesion, migration, and immune response. It is also closely related with the occurrence and development of many critical diseases. Therefore, large scale identification of protein glycosylation not only provides important information for the study of basic biological mechanisms, but also is crucial for the discovery of new diagnostic biomarkers and therapeutic targets. Due to the low abundance of glycoprotein/glycopeptide in real biologi- cal samples, enrichment before mass spectrometry （MS） analysis is an essential step for achie- ving deep glycosylation site coverage. Lectin enrichment, as an effective method for glycopro- teins/glycopeptides enrichment, has been utilized widely in glycoproteomics research. To solve the problems of low lectin loading and limited enrichment efficiency of existing lectin functional materials, we prepared two kinds of new graphene oxide （GO） immobilized lectin. Besides good dispersion in aqueous solution as well as good chemical stability, GO has extremely large specific surface area and also carries high density of functional groups on its surface, which isespecially beneficial for achieving high lectin loading amount. As a result, lectin loading as high as 1.90 mg/mg was achieved for GO-lectin （ GO-ConA 2. 073 mg/mg, RSD = 1.0% ; GO-WGA 1. 908 mg/mg, RSD = 0.14%）. One milligram GO-lectin can adsorb more than 200 μg glycopro- tein each experiment in two weeks. The GO-lectin was successfully applied in glycoproteins/ glycopeptides enrichment with high efficiency and selectivity, indicating its good application potential in glycoproteomics research.

关 键 词：凝集素 氧化石墨烯 富集 糖蛋白 糖肽 

分 类 号：O658[理学—分析化学]