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

STEP (statistical test of equivalent pathways) analysis: A mass spectrometric method for carbohydrates and peptides

5886-589

Glycans and glycoproteins as specific biomarkers for cancer

Protein glycosylation and other post-translational modifications are involved in potentially all aspects of human growth and development. Defective glycosylation has adverse effects on human physiological conditions and accompanies many chronic and infectious diseases. Altered glycosylation can occur at the onset and/or during tumor progression. Identifying these changes at early disease stages may aid in making decisions regarding treatments as early intervention can greatly enhance survival. This review highlights some of the efforts being made to identify N- and O-glycosylation profile shifts in cancer using mass spectrometry. The analysis of single or panels of potential glycoprotein cancer markers are covered. Other emerging technologies like global glycan release and site-specific glycosylation analysis and quantitation are also discussed

Reversed-phase separation methods for glycan analysis

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. [Figure not available: see fulltext.