Purification of N- and O-glycans and their derivatives from biological samples by the absorbent cotton hydrophilic chromatographic column

Abstract

Mass spectrum (MS) is one of the most commonly used tools for qualitative and quantitative analysis of glycans. However, due to the complexity of biological samples and the low ionization efficiency of glycans, these need to be purified and derivatized prior to MS analysis. Existing purification strategies require a combination of multiple methods and are cumbersome to operate. Here, we propose a new method for the purification of glycoprotein N/O-glycans and their derivatives using a hand-packed absorbent cotton hydrophilic interaction chromatography column (HILIC). The method's reliability and applicability were verified by purifying N/O-glycans and the derivatives of standard glycoproteins, such as chicken albumin and porcine stomach mucin. Stable isotope labelling was used to compare the glycans’ recovery following different purification methods. Absorbent cotton HILIC was also successfully applied for the analysis of human serum and fetal bovine serum glycoprotein N-glycans. Finally, testing revealed high binding capacity (9 mg/g−1 maltohexaose/absorbent cotton) and good recovery (average recovery was 91.7%) of glycans. Compared with traditional procedures, the proposed purification method offers considerable advantages, such as simplicity, high efficiency, economy, universality, and broad applicability for the pretreatment of glycans and their derivatives in biological samples prior to MS analysis.