Sialic Acid GlycoAnalysis

sialic acid analysis image

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
  • research

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)
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