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Guide to Glycosylation Analysis

Glycan Release

Liberation of glycoprotein-bound glycans by hydrazinolysis
or using enzymes

Glycan Labeling

Tagging of glycans with fluorescent or uv-active labels

Glycan Purification

Purification of glycans at various stages during analysis

Glycan HPLC

Preparation and analysis of glycans by HPLC



Glycan
Release
Glycan
Labeling
Glycan
Purification
Glycan
HPLC

Glycan Release

The first step in glycan analysis of glyconjugates such as glycoproteins and glycolipids is the
release of thesugars from the molecules to which they are attached.

Great care should be taken in choosing the best glycan release method from the wide range
of chemical and enzymatic techniques available. The main glycan release methods we use at
Ludger are hydrazinolysis and englycosidase treatment

· Hydrazinolysis
 

This is suitable for releasing both N- and O-linked glycans from glycoproteins. It involves the
incubation of the dried glycoprotein with anhydrous hydrazine followed by a straightforward
workup to purify the released glycans. For success, hydrazinolysis must be carried out with
absolutely pure hydrazine under strictly controlled conditions. Advantages of hydrazinolysis
include its versatility with the ability to release both N- and O-linked glycans in a virtually
non-selective way.

· Endoglycosidase Treatment

 

Endoglycosidase release is generally a simpler method to implement than hydrazinolysis
and suitable for many applications. There are a number of useful endoglycosidases including
PNGase F which releases most N-glycans from glycoproteins. Take care when using
endoglycosidases as there are a number of conditions and substances that can lead to
selective non-release of glycans. In particular, PNGase F does not release certain types of
N-glycan which have core fucose attached in the alpha1-3 position (e.g. plants) and it can
also have low activity against glycans which are found at positions near to either the C- or
N-terminii of the peptide backbone.

· Ludger Glycan Release Kits

Ludger produces kits for release of glycans by hydrazinolysis (cat no. LL-HYDRAZ-A2) and
for removing O-glycans (cat no. LL-ORELA-A2).

 
Glycan
Release
Glycan
Labeling
Glycan
Purification
Glycan
HPLC

Glycan Labeling

Release of a glycan by hydrazinolysis or endoglycosidase treatment produces a reducing terminus (the bit that was formerly attached to the protein or lipid) that can be tagged with
a fluorophore or chromophore label

· Which Glycan Label to Use?

 

There are a number of such labels available and the one to choose depends on :
 
· The physico-chemical properties of the glycans of interest - e.g. molecular size, the
  presence of charged groups such as sialic acids.

· The information required from the analysis - e.g. the degree of detail needed for
  quantitation, monosaccharide identity, linkage information, etc
· The analytical methods to be used. The variants include HPLC
  (including anion exchange, hydrophilic interaction, hydophobic interaction),
  detection by fluorescence, UV-absorbance, and amperometry, mass spectroscopy
  (including MALDI TOF and electrospray), gel electrophoresis, and TLC.
·Physical and chemical treatments to use. These include enzymatic degradation and
  synthesis, acid hydrolysis, drying methods, dissolution in different solvents
· Labeling Protoco
 

 

Most glycan labels are aromatic amines which are conjugated to the glycans by reductive
amination.
The procedure for this is outlined in our overview of the glycan labeling protocol

· Recommended Glycan Labels

Currently, there is no glycan label which excels for all applications. However, the following
are those that we find the most useful:

 
·2-AA (2-aminobenzoicacid) is a versatile fluorescent tag for glycans and is suitable for a
  wide range of applications including monosaccharide analysis, profiling or fingerprinting
  of complex glycosylation patterns by HPLC, and rapid analysis by gel electrophoresis.

·AA-Ac (3-(acetylamino)-6-aminoacridine) is a relatively new glycan probe that is proving
  very useful for rapid and sensitive profiling and structural analysis of glycans by HPLC
  and LC/MS
·2-AB is a versatile fluorescent tag for glycans and is suitable for a wide range of
  applications, profiling or fingerprinting of complex glycosylation patterns by anion
  exchange and reverse phase HPLC
·APTS - this label is commonly used for CE
 

Ludger provides labeling kits for the most useful of the glycan labels including those for
2-AA and AA-Ac

 
Glycan
Release
Glycan
Labeling
Glycan
Purification
Glycan
HPLC

Glycan Purification

Proper cleanup of glycans from contaminating substances such as salts, proteins, peptides,
or excess derivatization reagents is essential for the success of many treatments and
analyses such as mass spectrometry

 
Glycan cleanup methods that are particularly useful during glycosylation analysis include the
following:

 
·Hydrophilic interaction cleanup
  This involves application of the glycan to certain types of hydrophilic matrix
  (e.g. LudgerClean S cartridges) in the presence of high levels of particular
  organic solvents. The glycans bind to the matrix and hydrophobic non-glycan
  contaminants are washed off with the solvent. The purified glycans are
  subsequently recovered by elution with an aqueous solvent.

·Hydrophilic interaction cleanup
  This uses a special resin (e.g. LudgerClean EB10 cartridges) with a very flat
  (on a molecular) surface of sp2 hybridized electrons. The glycans are applied
  to the resin in an aqueous solution and are captured. Non-organic substances
  such as salts are washed away and the glycans are then recovered by elution
  using a mixed organic-aqueous solvent. The method works very well for a wide
  range of glycans and allows very rapid purification of the carbohydrate.
 
 
Glycan
Release
Glycan
Labeling
Glycan
Purification
Glycan
HPLC

Glycan HPLC

Fluorophore or chromophore labeled glycans can be analysed by a range of HPLC mthods
including hydrophilic interaction chromatography, electronic interaction, ion-exchange,
hydrophobic interaction and size-exclusion chromatography

 
Hydrophilic interaction HPLC is particularly useful for profiling or fingerprinting of complex
glycan mixtures and allows separation of a wide variety of glycan types. The principle is
similar to that for hydrophilic interaction cleanup (descried above) except that there is a
gradual change in eluant from high to low content of organic solvent. The various glycan
species are separated during this gradient and are typically followed by fluorescence
detection as they elute from the column. The LudgerSep N1 Amide HPLC column is suitable
for hydrophilic interaction HPLC of most N- and O-linked glycans. Good fluorescent tags to
use with the column include 2-AA (2-aminobenzoic acid)
 
The LudgerSepN2 HPLC column is used to perform hydrophilic interaction normal pahse
chromatography. 2AB or 2AA glycans are separated according to size and branching of
the glycans, with smaller glycans eluting first. The fluorescent peaks are compared to a
2AB/AA glucose homopolymer standard using a cubic spline calculation in order to obtain
a glucose unit (GU) values for each peak.