Mittermayr Stefan, Albrecht Simone, Váradi Csaba, Millán-Martín Silvia, Bones Jonathan
NIBRT-The National Institute for Bioprocessing Research & Training, Foster Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland.
Methods Mol Biol. 2017;1606:353-366. doi: 10.1007/978-1-4939-6990-6_23.
Liquid phase separation analysis and subsequent quantitation remains a challenging task for protein-derived oligosaccharides due to their inherent structural complexity and diversity. Incomplete resolution or co-detection of multiple glycan species complicates peak area-based quantitation and associated statistical analysis when optical detection methods are used. The approach outlined herein describes the utilization of stable isotope variants of commonly used fluorescent tags that allow for mass-based glycan identification and relative quantitation following separation by liquid chromatography (LC) or capillary electrophoresis (CE). Comparability assessment of glycoprotein-derived oligosaccharides is performed by derivatization with commercially available isotope variants of 2-aminobenzoic acid or aniline and analysis by LC- and CE-mass spectrometry. Quantitative information is attained from the extracted ion chromatogram/electropherogram ratios generated from the light and heavy isotope clusters.
由于蛋白质衍生的寡糖具有固有的结构复杂性和多样性，液相分离分析及后续定量仍然是一项具有挑战性的任务。当使用光学检测方法时，多种聚糖种类的不完全分离或共同检测会使基于峰面积的定量及相关统计分析变得复杂。本文概述的方法描述了利用常用荧光标签的稳定同位素变体，通过液相色谱（LC）或毛细管电泳（CE）分离后进行基于质量的聚糖鉴定和相对定量。通过用市售的2-氨基苯甲酸或苯胺的同位素变体进行衍生化，并通过LC-质谱和CE-质谱分析来进行糖蛋白衍生寡糖的可比性评估。定量信息从由轻、重同位素簇产生的提取离子色谱图/电泳图比率中获得。
