Liquid chromatography-selected reaction monitoring (LC-SRM) approach for the separation and quantitation of sialylated N-glycans linkage isomers.

Abstract

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.

• Authors: Shujuan Tao, Yining Huang, Barry E Boyes, Ron Orlando
• Journal: Analytical chemistry (Anal Chem) Vol. 86 Issue 21 Pg. 10584-90 (Nov 04 2014) ISSN: 1520-6882 [Electronic] United States
• PMID: 25299151 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Fetuins
• Polysaccharides
• Sialic Acids

Topics

• Animals
• Carbohydrate Sequence
• Cattle
• Chromatography, Liquid (methods)
• Fetuins (chemistry, isolation & purification)
• Humans
• Isomerism
• Mass Spectrometry (methods)
• Molecular Sequence Data
• Polysaccharides (chemistry, isolation & purification)
• Serum (chemistry)
• Sialic Acids (analysis)