Analysis of procainamide-derivatised heparan sulphate disaccharides in biological samples using hydrophilic interaction liquid chromatography mass spectrometry

Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6–4.9 ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA–GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biolog

Glycan labeling strategies and their use in identification and quantification

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

Glycomics using mass spectrometry

Mass spectrometry plays an increasingly important role in structural glycomics. This review provides an overview on currently used mass spectrometric approaches such as the characterization of glycans, the analysis of glycopeptides obtained by proteolytic cleavage of proteins and the analysis of glycosphingolipids. The given examples are demonstrating the application of mass spectrometry to study glycosylation changes associated with congenital disorders of glycosylation, lysosomal storage diseases, autoimmune diseases and cancer