人尿液蛋白质N-糖肽富集和质谱鉴定的新方法研究  

New Method for Human Urinary N-glycopeptide Enrichment and Identification by Mass Spectrometry

作　　者：黄俊杰 焦丰龙 王和平 朱丽华 秦伟捷[2] 钱小红[2] 

机构地区：[1]华北理工大学基础医学院,河北唐山063200 [2]军事医学科学院放射与辐射医学研究所,国家蛋白质科学中心(北京),蛋白质组学国家重点实验室,北京蛋白质组研究中心,北京102206 [3]河北省慢性疾病重点实验室,河北唐山063000

出　　处：《质谱学报》2018年第2期149-157,共9页Journal of Chinese Mass Spectrometry Society

基　　金：国家自然科学基金(21235001、21275005、21405175); 国家重点研发计划(2013CB911204、2016YFA0501403)资助

摘　　要：蛋白质的N-糖基化修饰参与了许多重要的生理过程,是研究多种重大疾病诊断标志物的热点。与其他体液活检样本相比,尿液可以无创、大量获取,并且不受稳态调节的影响,能够在一定程度上反映整个机体的生理和病理状态。因此,人尿液蛋白质N-糖基化的规模化研究对疾病诊断标记物的筛选和治疗靶点的发现均具有重大意义。由于人尿液中的N-糖蛋白含量有限,修饰比例较低,在质谱分析时N-糖肽易被高丰度的非糖肽所掩盖,难以鉴定。因此,发展高效、高选择性的富集材料是实现尿蛋白N-糖基化深度覆盖的必要前提。本研究利用巯基-烯点击化学反应,使用[3-(甲基丙烯酰氨基)丙基]二甲基(3-硫代丙基)氢氧化铵内盐(SPP)制备了两性离子修饰亲水硅胶材料(SPP-SiO2)。该材料成功地应用于标准糖蛋白和健康人尿蛋白N-糖肽的富集和质谱检测,共鉴定了包含1 065个位点的633个尿液糖蛋白,比文献报道的鉴定规模提高了12.2%,证明了该亲水材料在尿液糖蛋白质组研究中的应用潜力。Glycosylation plays important role in cell-cell interaction,signal transductionand cancer immunology,which is currently a hotspot in biomarkers screening for many major human diseases.Though commonly used in biomarker studies,plasma proteome/glycoproteome is under strict homeostatic control,which makes it difficult for biomarker discovery.As the partial filtrate of blood,urine serves not only as an ideal source of biomarker for diseases diagnosis of the urogenital system,but also as a potential source of information on physiological conditions.Therefore,in-depth analysis of human urine glycoproteome is of great significance.However,due to the low abundance of N-glycoprotein in human urine,limited modification stoichiometry and microheterogeneity of N-glycosylation,it is a highly challenging task for N-glycoprotein/glycopeptide identification by mass spectrometry.Therefore,the development of efficient and selective enrichment method is a prerequisite for large-scale identification of urinary protein N-glycosylation.In this study,we synthesized a novel zwitterionic stationary phase with high hydrophilicity based on the＂thiol-ene＂click reaction between[3-（methacryloylamino）propyl]dimethyl（3-thiopropyl）ammonium hydroxide（SPP）and mercapto silica.SPP-silica exhibited great potential in the enrichment of N-glycopeptides,while keeping the merit of a facile and time-saving synthesis protocol.To evaluate the practicability of SPP-silica in the selective enrichment of N-glycopeptides,a tryptic digest of standard glycoprotein（human IgG）was employed as the testing sample.36 glycopeptides were captured and identified.SPP-silica showed remarkable selectivity for N-linked glycopeptides even in the presence of 10-fold excess tryptic digests of nonglycoprotein（BSA）.SPP-silica was successfully applied to the HILIC enrichment and mass spectrometry identification of urinary protein N-glycopeptide from healthy adults.The same sample was enriched by three batches of SPP-silica and analyzed by mass spectrometry.9

关 键 词：巯基-烯点击化学法 尿蛋白 N-糖肽 富集 质谱鉴定 

分 类 号：O657.63[理学—分析化学]