Liquid Chromatography-Selected Reaction Monitoring(LC-SRM) Approach for the Separation and Quantitation of SialylatedN-Glycans Linkage Isomers

摘要

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-glycanisomers differing in α2-3 and α2-6 linkages using a novelsuperficially porous particle (Fused-Core) Penta-HILIC (hydrophilicinteraction liquid chromatography) column. SRM detection providesthe relative quantitation of each SA linkage isomer, and minimizesinterferences from coeluting glycans that are problematic for UV/Fluorescencebased quantitation. With our approach, the relative quantitation ofeach SA linkage isomer is obtained from a straightforward liquid chromatography-massspectrometry (LC-MS) experiment.