Tao Shujuan, Huang Yining, Boyes Barry E, Orlando Ron
Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
Anal Chem. 2014 Nov 4;86(21):10584-90. doi: 10.1021/ac5020996. Epub 2014 Oct 27.
The study of N-linked glycans is among the most challenging bioanalytical tasks because of their complexity and variety. The presence of glycoform families that differ only in branching and/or linkage position makes the identification and quantitation of individual glycans exceedingly difficult. Quantitation of these individual glycans is important because changes in the abundance of these isomers are often associated with significant biomedical events. For instance, previous studies have shown that the ratio of α2-3 to α2-6 linked sialic acid (SA) plays an important role in cancer biology. Consequently, quantitative methods to detect alterations in the ratios of glycans based on their SA linkages could serve as a diagnostic tool in oncology, yet traditional glycomic profiling cannot readily differentiate between these linkage isomers. Here, we present a liquid chromatography-selected reaction monitoring (LC-SRM) approach that we demonstrate is capable of quantitating the individual SA linkage isomers. The LC method is capable of separating sialylated N-glycan isomers differing in α2-3 and α2-6 linkages using a novel superficially porous particle (Fused-Core) Penta-HILIC (hydrophilic interaction liquid chromatography) column. SRM detection provides the relative quantitation of each SA linkage isomer, and minimizes interferences from coeluting glycans that are problematic for UV/Fluorescence based quantitation. With our approach, the relative quantitation of each SA linkage isomer is obtained from a straightforward liquid chromatography-mass spectrometry (LC-MS) experiment.
由于其复杂性和多样性，N-连接聚糖的研究是最具挑战性的生物分析任务之一。仅在分支和/或连接位置上存在差异的糖型家族的存在，使得单个聚糖的鉴定和定量极其困难。这些单个聚糖的定量很重要，因为这些异构体丰度的变化通常与重大生物医学事件相关。例如，先前的研究表明，α2-3连接唾液酸（SA）与α2-6连接唾液酸的比例在癌症生物学中起着重要作用。因此，基于SA连接来检测聚糖比例变化的定量方法可以作为肿瘤学中的诊断工具，但传统的糖组学分析方法无法轻易区分这些连接异构体。在此，我们提出了一种液相色谱-选择反应监测（LC-SRM）方法，我们证明该方法能够对单个SA连接异构体进行定量。该液相色谱方法能够使用新型表面多孔颗粒（熔融核）五氟苯基亲水作用液相色谱（Penta-HILIC）柱分离α2-3和α2-6连接不同的唾液酸化N-聚糖异构体。SRM检测提供了每个SA连接异构体的相对定量，并最大限度地减少了基于紫外/荧光定量时共洗脱聚糖产生的干扰。通过我们的方法，每个SA连接异构体的相对定量可通过直接的液相色谱-质谱（LC-MS）实验获得。
