Further analysis by MALDI-TOF MS was performed after treatment overnight with either β-galactosidase (recombinant Aspergillus niger lacA; prepared in-house (38 (link))), α-fucosidase (bovine kidney from Sigma-Aldrich), α-mannosidase (Xanthomonas manihotis α1,2/3-specific from NEB) or β-N-acetylhexosaminidase (recombinant Apis mellifera FDL; prepared in-house, specific for the N-acetylglucosamine attached to core α1,3-mannose, i.e., the residue transferred by GlcNAc-TI (39 )) in 25 mM ammonium acetate, pH 4.5, at 37 °C. For removal of α1,3-linked fucose or of phosphorylcholine, selected fractions were dried and incubated overnight at 0 °C with 3 μl 48% (v/v) hydrofluoric acid prior to evaporation; the samples were diluted in water and re-evaporated, prior to redissolving once again.
Protocol detail
Glycan Analysis by MALDI-TOF MS
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Acetylglucosamine
After treatment
Ammonium acetate
Apis
Aspergillus niger
Bovine
Fucose
Fucosidase
Glcnac ti
Hydrofluoric acid
Kidney
Mannose
Mannosidase
Ms maldi
Phosphorylcholine
Xanthomonas
β galactosidase
Corresponding Organization :
Other organizations : BOKU University
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Variable analysis
independent variables
- Treatment overnight with β-galactosidase (recombinant Aspergillus niger lacA; prepared in-house (38 (link)))
- Treatment overnight with α-fucosidase (bovine kidney from Sigma-Aldrich)
- Treatment overnight with α-mannosidase (Xanthomonas manihotis α1,2/3-specific from NEB)
- Treatment overnight with β-N-acetylhexosaminidase (recombinant Apis mellifera FDL; prepared in-house, specific for the N-acetylglucosamine attached to core α1,3-mannose, i.e., the residue transferred by GlcNAc-TI (39 (no link found)))
- MALDI-TOF MS analysis
- 25 mM ammonium acetate, pH 4.5, at 37 °C
- Overnight incubation at 0 °C with 3 μl 48% (v/v) hydrofluoric acid for removal of α1,3-linked fucose or of phosphorylcholine
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