Jiang Kuan, Zhu He, Xiao Cong, Liu Ding, Edmunds Garrett, Wen Liuqing, Ma Cheng, Li Jing, Wang Peng George
State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300353, China; Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States.
Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States.
Anal Chim Acta. 2017 Apr 15;962:32-40. doi: 10.1016/j.aca.2017.01.049. Epub 2017 Jan 31.
Reductive amination is an indispensable method for glycomic analysis, as it tremendously facilitates glycan characterization and quantification by coupling functional tags at the reducing ends of glycans. However, traditional in-solution derivatization based approach for the preparation of reductively aminated glycans is quite tedious and time-consuming. Here, a simpler and more efficient strategy termed solid-phase reductive amination was investigated. The general concept underlying this new approach is to streamline glycan extraction, derivatization, and purification on non-porous graphitized carbon sorbents. Neutral and sialylated standard glycans were utilized to test the feasibility of the solid-phase method. As results, almost complete labeling of those glycans with four common labels of aniline, 2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA) and 2-amino-N-(2-aminoethyl)-benzamide (AEAB) was obtained, and negligible desialylation occurred during sample preparation. The labeled glycans derived from glycoproteins showed excellent reproducibility in high performance liquid chromatography (HPLC) and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Direct comparisons based on fluorescent absorbance and relative quantification using isotopic labeling demonstrated that the solid-phase strategy enabled 20-30% increase in sample recovery. In short, the solid-phase strategy is simple, reproducible, efficient, and sensitive for glycan analysis. This method was also successfully applied for N-glycan profiling of HEK 293 cells with MALDI-TOF MS, showing its attractive application in the high-throughput analysis of mammalian glycome.
还原胺化是糖组学分析中不可或缺的方法,因为它通过在聚糖的还原端连接功能标签极大地促进了聚糖的表征和定量。然而,传统的基于溶液中衍生化的还原胺化聚糖制备方法相当繁琐且耗时。在此,研究了一种更简单、更高效的策略,即固相还原胺化。这种新方法的基本概念是在无孔石墨化碳吸附剂上简化聚糖的提取、衍生化和纯化。利用中性和唾液酸化标准聚糖来测试固相方法的可行性。结果,用苯胺、2-氨基苯甲酰胺(2-AB)、2-氨基苯甲酸(2-AA)和2-氨基-N-(2-氨基乙基)-苯甲酰胺(AEAB)这四种常见标签对这些聚糖进行了几乎完全的标记,并且在样品制备过程中唾液酸的去唾液酸化可以忽略不计。从糖蛋白衍生的标记聚糖在高效液相色谱(HPLC)和基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)分析中显示出优异的重现性。基于荧光吸光度的直接比较和使用同位素标记的相对定量表明,固相策略使样品回收率提高了20-30%。简而言之,固相策略对于聚糖分析而言简单、可重现、高效且灵敏。该方法还成功应用于通过MALDI-TOF MS对HEK 293细胞进行N-聚糖谱分析,显示出其在哺乳动物糖组高通量分析中的诱人应用前景。
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