How can I release the N-linked glycans from my glycoprotein?

There are two main techniques commonly used to liberate glycoprotein N-linked glycans for glycosylation analysis - hydrazinolysis and endoglycosidase treatment. Our Guide to Glycosylation Analysis covers some of the considerations for choosing between these release methods.

How can I stop the sialic acids falling off my glycans?

Sialic acids are tricky things. In our experience, the purer the glycan sample the more likely the sialic acid residues are to fall off. There are some sensible precautions though:

  1. Avoid the combination of acidic pH and elevated temperature. Wherever possible, keep the pH in the range 6-9 and the temperature below 30 C.
  2. Be careful when drying glycan samples by centrifugal evaporation or rotary evaporation. When you use these procedures try to keep the temperature below 25 C. Lyophilization (freeze-drying) can be safer with respect to sialic acid degradation but with this technique there is a greater danger of losing the entire sample when the vacuum is released.

Which are the best fluorescent tags for glycan analysis?

The following table shows the most widely used fluorescent glycan tags for glycan analysis by HPLC, CE and MS:

Fluorescent Glycan Label Main Applications Fluorescence Wavelengths and Common Analysis Methods
Oligosaccharide Profiling by HPLC-FD and MS λex = 250 or 330 nm, λem = 428 nm

HPLC (amide, C18, AEX, HPAE-FD,
Proc (procainamide) Oligosaccharide Profiling by HPLC-FD and sensitive MS λex = 310 nm, λem = 370 nm

HPLC (amide, C18, AEX, PGC), ESI-MS
(2-aminobenzoic acid)
Oligosaccharide Profiling by HPLC-FD and MS
Monosaccharide Profiling by HPLC-FD

λex = 250nm or 360nm, λem = 425 nm

Oligosaccharides: HPLC (amide, C18, AEX, PGC), ESI-MS, MALDI-MS
Monosaccharides: HPLC (C18)
Oligosaccharide Profiling by CE λex = 455 - 488 nm, λem = 510 - 520 nm

VTag IgG Glycopeptide Profiling by HPLC-FD λex = 250 nm, λem = 360 nm

HPLC (amide), MALDI-MS
Profiling of Human vs Non-
Human Sialylation by HPLC
λex = 373 nm, λem = 448 nm

HPLC (C18)

Key: AEX = anion exchange, HPAE-FD = high pH anion exchange with fluorescence detection, PGC = porous graphitic carbon