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Monosaccharide analysis (mammalian)

General description

The scope of monosaccharide analysis is to obtain free monosaccharides released from N-glycans. Due to the distinct chemical properties of various monosaccharides, there are many different methodologies for monosaccharide analysis. Here, FGCZ focuses on the quantitative release of hexoses, pentoses, and hexosamines from glycoconjugates.

Workflow

The workflow contains four steps,

• 1. acid hydrolysis of released N-glycans
  • strong acid hydrolysis
    • Released N-glycans are treated with 2M TFA for 3 hours at 100°C and dried by SpeedVac.
• 2. derivatization
• 1. 2-Aminobenzoic acid (2AA) is used to label the reducing end of monosaccharides.
  • Resulting hydrolysates were reconstituted by 5μL of 80mg/mL sodium acetate trihydrate.
  • 2AA labelling solution was prepared by dissolving 30mg of 2AA in 1mL of 2% (w/v) boric acid methanol.
  • This suspension was then used to dissolve 30mg of sodium cyanoborohydride (NaBH3CN).
  • 10uL of this solution was added to each of the monosaccharide mixtures and the samples were incubated at 80°C for 60mins.
• 3. LC-FLD analysis
• The labeled monosaccharides are separated and detected by Waters H-Class HPLC-fluorescence detector (LC-FLD) and Excitation wavelength: 360nm; Emission wavelength: 425nm. Data are recorded by Masslynx 4.2

Reference

1. Manzi, A 1995 Current Protocols in Molecular biology, 17.16.1-17.16.11 Acid hydrolysis for release of monosaccharides
2. Stepan, H et al 2011 Analytical Biochemistry 418, 24-29 optimization of monosaccharide determination using anthranilic acid and 1-phenyl-3-methyl-5-pyrazolone for gastropod analysis
3. Waters application note Future Proofing the Biopharmaceutical QC Laboratory: Chromatographic Scaling of HPLC Monosaccharide Analyses Using the ACQUITY UPLC H-Class Bio System

Questions?

• Check our FAQs Glycan / Glycopeptide analysis page or contact us at proteomics at fgcz.ethz.ch
• If you are interested in other monosaccharides, please contact us at proteomics at fgcz.ethz.ch