Protein Glycosylation: Implications for In Vivo Functions and Therapeutic Applications - Advances in Biochemical Engineering Biotechnology - page 189

Table 4 (continued)

Analysis Techniques followed Remarks Ref.

C) Separation of glycans Chromatography

1. Normal phase HPLC Capable of resolving subpicomolar quantities of [265]

mixtures of fluorescently labelled neutral and
acidic N - and O -glycans simultaneously

2. RP-HPLC The effect of linkage position is much more [266]

marked on RP-separations than on normal phase.
Oligosaccharides containing bisecting Gn can be identified.
Derivatization (e.g. 2-AP for fluorescence
detection) to increase hydrophobicity required

3. Weak anion exchange HPLC Suitable for resolving mixture of glycans (above pH 12) [267]

on the basis of the number of charged residues. High pH AEC
coupled with pulsed amperometric detector (PAD)
is routinely used for separating and profiling of
sialylated N - and O -glycans

4. Gel permeation (BioGel P4) Applicable for neutral oligosaccharides. So sample desialylation

is essential before use. It allows sequencing of
glycans, if associated with the treatment of exoglycosidases

5. Lectin affinity chromatography Used in techniques such as immobilized lectin affinity [268]

chromatography

Capillary Electrophoresis

Used to separate glycopeptides and released glycan chains [269,

with UV- or laser-induced fluorescence (LIF) detection. 270]

On of the few techniques able to resolve glycoforms. Suitable
as a rapid fingerprinting technique for assessing the
glycosylation varients. Oligomannose, hybrid and complex
sialylated oligo-saccharides can be resolved

Structural analysis (applicable to the alternate approaches):
1. Digestion of glycopeptides/ Digestion of each oligosaccharide with enzymes, [271]

glycans with highly specific either sequentially or using enzyme arrays. Extremely

exoglycosidases sensitive and provides information on all parameters