This module provides relative or absolute quantitative information on the neutral (i.e. non-anionic) monosaccharide composition: GlcN, GalN, Gal, Man, Glc, Fuc, Xyl.
The analysis will be performed on single or triplicate releases of samples (typically 50-200 µg for each release), an equivalent amount (by volume) of sample buffer negative control, and alongside Ludger’s system suitability standards, positive (including Ludger’s quantitative glycopeptide standard) and negative controls.
This module is suitable for:
- quality control
- monitoring batch to batch consistency
- comparability studies
Quantitative Monosaccharide Analysis
Neutral sugars are hydrolyzed by incubation with trifluoroacetic acid (TFA)* and/or hydrochloric acid (HCl)*, labelled with 2-AA and analyzed by LudgerSep R2 HPLC with fluorescence detection.
The absolute amounts of GlcN, GalN, Gal, Man, Glc, Fuc, are calculated by reference to standard curves from Ludger’s quantitative standard containing 6 monosaccharides (GlcN, GalN, Gal, Man, Glc, Fuc). The presence and relative quantitation of Xyl is determined by comparison to Xyl standard.
*The 2M TFA will release all of the monosaccharides. If harsher conditions are required (as can be the case to completely remove the core N-acetylhexosamines which are directly attached to the protein) 6M HCL can be used, however there will be degradation of the hexose sugars under these conditions, therefore release conditions may require optimising for individual glycoproteins.
Final report contains:
- HPLC profiles for system suitability standards and Ludger positive controls
- HPLC profiles for client samples and buffer negative controls
- Quantitative data for GlcNAc, GalNAc, Gal, Man, Glc and Fuc as nmol/mg protein
- Quantitative data for Xyl as nmol/mg protein