Reversed-phase separation methods for glycan analysis

Abstract

Reversed-phase chromatography is a method that is often used for glycan separation. For this, glycans are often derivatized with a hydrophobic tag to achieve retention on hydrophobic stationary phases. The separation and elution order of glycans in reversed-phase chromatography is highly dependent on the hydrophobicity of the tag and the contribution of the glycan itself to the retention. The contribution of the different monosaccharides to the retention strongly depends on the position and linkage, and isomer separation may be achieved. The influence of sialic acids and fucoses on the retention of glycans is still incompletely understood and deserves further study. Analysis of complex samples may come with incomplete separation of glycan species, thereby complicating reversed-phase chromatography with fluorescence or UV detection, whereas coupling with mass spectrometry detection allows the resolution of complex mixtures. Depending on the column properties, eluents, and run time, separation of isomeric and isobaric structures can be accomplished with reversed-phase chromatography. Alternatively, porous graphitized carbon chromatography and hydrophilic interaction liquid chromatography are also able to separate isomeric and isobaric structures, generally without the necessity of glycan labeling. Hydrophilic interaction liquid chromatography, porous graphitized carbon chromatography, and reversed-phase chromatography all serve different research purposes and thus can be used for different research questions. A great advantage of reversed-phase chromatography is its broad distribution as it is used in virtually every bioanalytical research laboratory, making it an attracting platform for glycan analysis. Graphical Abstract Glycan isomer separation by reversed phase liquid chromatography.

Keywords: Glycan; Liquid chromatography; Reversed phase; Separation.

Graphical Abstract

Glycan isomer separation by reversed phase liquid chromatography

Fig. 1

Structures of labels used in reversed-phase chromatography of oligosaccharides. AA anthranilic acid, AA-Ac 3-(acetylamino)-6-aminoacridine, AB 2-aminobenzamide, ABBE 4-aminobenzoic acid butyl ester, ABEE 4-aminobenzoic acid ethyl ester, ABME >4-aminobenzoic acid methyl ester, ABP 2-amino-5-bromopyridine, AMAC 2-aminoacridone, ANTS 2-aminonapthalene trisulfone, HOA 4-n-heptyloxyaniline, INLIGHT individuality normalization when labeling with isotopic glycan hydrazide tags, PA 2-aminopyridine, PMP 1-phenyl-3-methyl-5-pyrazolone

Fig. 2

a Overview of the chromatograms of various reducing end derivatized N-glycans and a native glycan that is eluted at the void volume (dashed line). b Reversed-phase chromatograms of desialylated immunoglobulin G N-glycans derivatized with PA, AB, or ABEE. c Chromatograms of the separation of a mixture of four glycans: (GlcNAc)₂(Man)₃(GlcNAc)₂, (GlcNAc)₂(Man)₃, and two isomers of (GlcNAc)₂(Man)₃(GlcNAc), where GlcNAc is N-acetylglucosamine and Man is mannose. RP reversed phase. (Reproduced and modified from Pabst et al. [70] with permission)