Guide to Glycosylation Analysis

Glycan Release

The first step in glycan analysis of glyconjugates such as glycoproteins and glycolipids is the release of the sugars 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).

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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 Protocol

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-aminobenzoic acid) 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.

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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.
  • Electronic 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.

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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.

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