Purification of Derivatized Oligosaccharides by Solid Phase Extraction for Glycomic Analysis

Qiwei Zhang,Xin Liu,Xiaojun Feng,Bi-Feng Liu,Henghui Li,Nikos K Karamanos

doi:10.1371/journal.pone.0094232

Qiwei Zhang, Xin Liu + Show 4 more

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https://doi.org/10.1371/journal.pone.0094232

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Abstract

Profiling of glycans released from proteins is very complex and important. To enhance the detection sensitivity, chemical derivatization is required for the analysis of carbohydrates. Due to the interference of excess reagents, a simple and reliable purification method is usually necessary for the derivatized oligosaccharides. Various SPE based methods have been applied for the clean-up process. To demonstrate the differences among these methods, seven types of self-packed SPE cartridges were systematically compared in this study. The optimized conditions were determined for each type of cartridge and it was found that microcrystalline cellulose was the most appropriate SPE material for the purification of derivatized oligosaccharide. Normal phase HPLC analysis of the derivatized maltoheptaose was realized with a detection limit of 0.12 pmol (S N−1 = 3) and a recovery over 70%. With the optimized SPE method, relative quantification analysis of N-glycans from model glycoproteins were carried out accurately and over 40 N-glycans from human serum samples were determined regardless of the isomers. Due to the high stability and sensitivity, microcrystalline cellulose cartridge showed potential applications in glycomics analysis.

Highlights

Glycosylation represents one of the most complex and widespread post-translational modifications of human proteins
The aim of this study is to find out the most appropriate hydrophilic interaction chromatography (HILIC) solid-phase extraction (SPE) method and to establish a standard strategy for the purification of labeled oligosaccharides
For microcrystalline cellulose (MCC), literature has shown that a small amount of acid in the washing solution could enhance the efficiency in the enrichment of glycopeptides [31]

Summary

Introduction

Glycosylation represents one of the most complex and widespread post-translational modifications of human proteins. Aberrant glycosylation profile may lead to dramatic changes of the proteins in the activity, distribution as well as stability [4,5,6,7]. To enhance the detection sensitivity, oligosaccharides are usually derivatized prior to analysis. Most of such derivatization reactions can be accomplished by coupling the reagents with the reducing end of saccharides [17,19]. During this step, reagents, including salts, derivatization reagents and solvent are normally presented in large excess. A clean-up procedure has to be implemented to remove the excess reagents that often interfere with the following detection [20]

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