Most methods for the analysis of oligosaccharides from biological sources require a glycan derivatization step: glycans may be derivatized to introduce a chromophore or fluorophore, facilitating detection after chromatographic or electrophoretic separation. Derivatization can also be applied to link charged or hydrophobic groups at the reducing end to enhance glycan separation and mass-spectrometric detection. Moreover, derivatization steps such as permethylation aim at stabilizing sialic acid residues, enhancing mass-spectrometric sensitivity, and supporting detailed structural characterization by (tandem) mass spectrometry. Finally, many glycan labels serve as a linker for oligosaccharide attachment to surfaces or carrier proteins, thereby allowing interaction studies with carbohydrate-binding proteins. In this review, various aspects of glycan labeling, separation, and detection strategies are discussed.FigureMALDI-FTICR-MS of 2AA-labeled total plasma N-glycans

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Many lysosomal storage diseases are characterized by an increased urinary excretion of glycoconjugates and oligosaccharides that are characteristic for the underlying enzymatic defect. Here, we have used capillary high-performance anion-exchange chromatography (HPAEC) hyphenated to mass spectrometry to analyze free oligosaccharides from urine samples of patients suffering from the lysosomal storage disorders fucosidosis, α-mannosidosis, GM1-gangliosidosis, GM2-gangliosidosis, and sialidosis. Glycan fingerprints were registered, and the patterns of accumulated oligosaccharides were found to reflect the specific blockages of the catabolic pathway. Our analytical approach allowed structural analysis of the excreted oligosaccharides and revealed several previously unpublished oligosaccharides. In conclusion, using online coupling of HPAEC with mass spectrometric detection, our study provides characteristic urinary oligosaccharide fingerprints with diagnostic potential for lysosomal storage disorders.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-012-5968-9) contains supplementary material, which is available to authorized users.

High-performance anion-exchange chromatography (HPAEC) coupled to pulsed amperometric detection (PAD) is a highly sensitive method for the analysis of oligosaccharides without the need for prior derivatization. However, the method suffers from the lack of chemical information with peak assignments based on the retention times of authentic standards or known peaks of reference materials. Here we applied HPAEC coupled on-line with electrospray ion trap mass spectrometry (HPAEC-MS) using a prototype mini-bore (1mm I.D.) CarboPac PA200 column and challenged the analytical separation based method for the structural assignment of heterogeneous mixtures of N-glycans derived from immunoglobulin G from human plasma, glyco-engineered CHO cells, and Sp2/0 mouse myeloma cells. Compared to an analytical scale 3mm I.D. column, the mini-bore column demonstrated a superior performance with up to 8-fold improved limit of detection for specific N-glycans determined by PAD. Quantitative evaluation by extracted ion current chromatograms revealed detection limits in the 50-100 femtomole range using ion trap MS operated in positive ionization mode. In our hands HPAEC-MS/MS allowed the detection and quantification of even low abundant glycan species including biantennary complex-type, high mannose, hybrid and hybrid bisected structures. In comparison to the detection of N-glycans as lithiated or sodiated adducts, we obtained a 65-fold improved signal-to-noise ratio with protonated ions only. Relative quantitative evaluation by single ion current chromatograms was successfully applied and demonstrated an excellent performance with respect to selectivity in the relative quantification of heterogeneous samples of N-glycans compared to HPAEC-PAD and HILIC-UPLC of 2-AB labelled N-glycans.

A system for the determination of carbohydrates based on on‐line microdialysis introduction high‐performance anion‐exchange chromatography integrated pulsed electrochemical detection/mass spectrometry (MS) using ionspray as the ionization technique, with on‐line desalting, is presented. This coupling of techniques utilizes a cation‐exchange membrane desalting device (CEMDD) which permitted the exchange of sodium ions with hydronium ions when sodium ion concentrations up to 600 mM were used to separate and elute oligosaccharides. The effective on‐line desalting by the CEMDD of the effluent before its introduction into the mass spectrometer was achieved by the electrolysis of water with a 500 mA current. The use of the CEMDD in combination with ionspray eliminated the need for a booster pump and also the regenerating water improved the sensitivity in comparison with the use of sulphuric acid as oligosaccharides could be detected down to 3 μg ml‐1. The system was developed and used to monitor cationized oligosaccharides based on their singly [M+Na]+ and doubly sodiated [(M+2Na)/2]2+ molecules during the hydrolysis of wheat starch. The MS/MS properties of maltose, trehalose and sucrose were used in combination with retention times to identify these disaccharides based on their dissociation patterns, which are characteristic of the type of glycosidic linkage. © 1998 by John Wiley & Sons, Ltd.

Urine, amniotic fluid and ascitic fluid samples of galactosialidosis patients were analyzed and structurally characterized for free oligosaccharides using capillary high‐performance anion‐exchange chromatography with pulsed amperometric detection and online mass spectrometry. In addition to the expected endo‐β‐N‐acetylglucosaminidase‐cleaved products of complex‐type sialylated N‐glycans, O‐sulfated oligosaccharide moieties were detected. Moreover, novel carbohydrate moieties with reducing‐end hexose residues were detected. On the basis of structural features such as a hexose–N‐acetylhexosamine–hexose–hexose consensus sequence and di‐sialic acid units, these oligosaccharides are thought to represent, at least in part, glycan moieties of glycosphingolipids. In addition, C1‐oxidized, aldohexonic acid‐containing versions of most of these oligosaccharides were observed. These observations suggest an alternative catabolism of glycosphingolipids in galactosialidosis patients: oligosaccharide moieties from glycosphingolipids would be released by a hitherto unknown ceramide glycanase activity. The results show the potential and versatility of the analytical approach for structural characterization of oligosaccharides in various body fluids.

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