This module provides absolute amounts of Neu5Ac and Neu5Gc and relative amounts of Neu5,9Ac2. 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 sialylated glycopeptide standard BQ-GPEP-A2G2S2-10U) and negative controls.
This module is suitable for:
- sialylated samples
- quality control
- monitoring batch to batch consistency
- comparability studies
In order to gain more information on relative proportion of the mono-, di-, tri- and tetra-sialylated glycan structures present in the samples WAX-HPLC module is required.
Quantitative Sialic Acid Release and DMB Labelling
Sialic acids are released from glycoproteins by mild acid hydrolysis, labelled with 1,2-diamino-4,5-methylenedioxybenzene (DMB) and analysed by LudgerSep R1 HPLC with fluorescence detection.
The absolute amounts of Neu5Ac & Neu5Gc are calculated by reference to standard curves from Ludger’s quantitative Neu5Ac & Neu5Gc standards. The presence of O-acetylated sialic acids is determined by comparison to a Neu5,9Ac2 standard and to a sialic acid reference panel containing Neu5Ac, Neu5Gc, Neu5,7Ac2, Neu5,Gc9Ac, Neu5,9Ac2 and Neu5,7,(8),9Ac3Gc. The relative proportions of these different sialic acids are determined from the peak areas.
Final report contains:
- HPLC profiles for system suitability standards and Ludger positive controls
- HPLC profiles for client samples and buffer negative controls
- Quantitation of Neu5Ac & Neu5Gc as nmoles/mg protein
- Relative quantitation of the O-acetylated sialic acids (e.g. Neu5,9Ac2)