Glycomics methods and tools

• N-glycans can be enzymatically released PNGase F (Glycerol-free), Recombinant after which they can be derivatized by premethylation or fluorescence labeling (via reductive amination).
• O-glycans can be chemically released (beta elimination, hydrazinolysis), converted to their alditols, or derivatized by permethylation.
• Permethylation allows efficient ionization and the formation of fragments during collision-induced-dissociation (CID). Permethylation of isolated glycans facilitates tandem MS experiments, where detailed structural information can be obtained.
• Alternatively, glycans can be labeled with fluorescent tags for sensitive detection and quantitation during liquid chromatography coupled with fluorescence detection (LC-FLD). These groups might also assist ionization for mass spectrometry (MS), or bear charges for capillary electrophoresis (CE).
• Commonly used tags are 2-aminobenzamide (2AB), 2-aminobenzoic acid (2AA), procainamide (PCA), and 8-aminopyrene-1,3,6-trisulfonic acid (APTS, used in CE). These tags are introduced via reductive amination in the presence of an acid catalyst and a reducing agent.
• Detailed structural analysis of released N-glycans is possible by a combination of LC-MS analysis, which identifies N-glycans by retention time and exact mass, coupled with exoglycosidase array analysis (N-Glycan Sequencing Kit E0577s).
• O-glycan alditols can be also sequenced using exoglycosidase analysis.

References: 

1. Mulloy, B., et al. (2017) Essentials of Glycobiology, Chapter 50. Structural Analysis of Glycans. PMID 28876844
2. Muchena, J. et al. (2014) Anal. Chem. 86(1): 196 - 212. PMID 24313268
3. Reinhold, V. et al. (2013) Mol. Cell Proteomics. 12(4):866-73. PMID 23438731