反相液相色谱-电喷雾-离子阱-飞行时间质谱法定量分析N-非取代肝素/硫酸乙酰肝素  

Quantitative Analysis of N-unsubstituted Heparin/Heparan Sulfate by Reversed-phase Liquid Chromatography-Electrospray Ionization Ion Trap-Time-of-Flight Mass Spectrometry

作　　者：杜佳燕 黄海月 苏晓明 魏峥[2] DU Jia-yan;HUANG Hai-yue;SU Xiao-ming;WEI Zheng(College of Chemistry, Fuzhou University, Fuzhou 350002, China;Institute of Glycobiochemistry, National Engineering Research Centre of Chemical Fertilizer Catalyst, Fuzhou University, Fuzhou 350002, China)

机构地区：[1]福州大学化学学院,福建福州350002 [2]福州大学化肥催化剂国家工程研究中心糖生化研究所,福建福州350002

出　　处：《质谱学报》2019年第3期222-232,I0001,共12页Journal of Chinese Mass Spectrometry Society

基　　金：国家自然科学基金(21343013)资助

摘　　要：肝素和硫酸乙酰肝素由重复的二糖单元组成,其组分和结构与重要的生物病理、生理作用息息相关。这些二糖单元包括含量较高的N-硫酸化葡萄糖胺、N-乙酰化葡萄糖胺和含量较低的N-非取代葡萄糖胺残基。本研究采用2-氨基吖啶酮标记二糖,结合反相液相色谱-电喷雾-离子阱-飞行时间质谱法(RP-LC-ESI-IT-TOFMS)分析肝素/硫酸乙酰肝素中N-非取代二糖。通过优化检测条件,实现了12种肝素/硫酸乙酰肝素二糖的基线分离,并采用外标法相对定量分析各二糖组分。该方法适用于分析含有N-非取代二糖的肝素衍生物,可为进一步研究N-非取代葡萄糖胺的结构与功能提供检测方法,有助于更好地理解N-非取代葡萄糖胺残基在人类健康与疾病管理中的重要作用。Heparan sulfate (HS) and heparin are complex sulfated glycosaminoglycans which have important biological activities in developmental processes, angiogenesis, blood coagulation, cell adhesion, and tumor metastasis. These interactions involve a wide variety of proteins, i.e. enzymes, cytokines, growth factors, extracellular matrix proteins. HS and heparin consist of repeating disaccharide units each composed of an uronic acid (UA) or sulfated at C-2 (UA2S) and a derivative of glucosamine [ N -acetylglucosamine (GlcNAc), N -sulfated glucosamine (GlcNS), or unsubstituted glucosamine (GlcNH 3 +)] that is variably O sulfated. The characterization of GlcNH 3 +-containing HS/heparin is fundamental to further elucidating such functional properties. In this study, a method of reversed-phase liquid chromatography-electrospray ionization ion trap-time-of-flight mass spectrometry (RP-LC-ESI-IT-TOF MS) was described for the separation and structural analysis of N -unsubstituted disaccharides from heparin/HS. Firstly, 12 disaccharides of heparin/HS were labeled with 2-aminoacridone ( AMAC ). Subsequently, the AMAC-labeled disaccharides were separated by RP-LC with varied reversed-phase columns. The results showed that the baseline separation of 12 AMAC-labeled heparin/HS disaccharides could be achieved by the ODS-2 HYPERSIL C18 column with a linear elution comprised by eluent A(40 mmol/L ammonium acetate solution (pH 5.6)) and eluent B (methanol). The mass spectrometer detector voltage was 1.8 kV, and the curved desolvation line and heat block temperatures both were 200 ℃. In mass spectrometry, both the ionization efficiency of each disaccharide, and the sulfate loss in the mass spectrometer ion source were different. In order to correct the quality deviation caused by above situation, the relative quantitative analysis of the 12 AMAC-labeled disaccharides was performed by calibration curves established by analyzing a series of increasing amounts of standard AMAC-labeled disaccharide mixtures(200-400 ng). Linearity was assess

关 键 词：硫酸乙酰肝素 N-非取代二糖 2-氨基吖啶酮 反相液相色谱-电喷雾-离子阱-飞行时间质谱(RP-LC-ESI-IT-TOFMS) 

分 类 号：O657.63[理学—分析化学]