The following are examples of the commonly used techniques in glycan analysis:
High-resolution mass spectrometry (MS) and high-performance liquid chromatography (HPLC) The most commonly applied methods are
MS and
HPLC, in which the glycan part is cleaved either enzymatically or chemically from the target and subjected to analysis. In case of glycolipids, they can be analyzed directly without separation of the lipid component. N-
glycans from glycoproteins are analyzed routinely by high-performance-liquid-chromatography (reversed phase, normal phase and ion exchange HPLC) after tagging the reducing end of the sugars with a fluorescent compound (reductive labeling). A large variety of different labels were introduced in the recent years, where 2-aminobenzamide (AB), anthranilic acid (AA), 2-aminopyridin (PA), 2-aminoacridone (AMAC) and 3-(acetylamino)-6-aminoacridine (AA-Ac) are just a few of them. O-
glycans are usually analysed without any tags, due to the chemical release conditions preventing them to be labeled. Fractionated glycans from
high-performance liquid chromatography (HPLC) instruments can be further analyzed by
MALDI-TOF-MS(MS) to get further information about structure and purity. Sometimes glycan pools are analyzed directly by
mass spectrometry without prefractionation, although a discrimination between isobaric glycan structures is more challenging or even not always possible. Anyway, direct
MALDI-TOF-MS analysis can lead to a fast and straightforward illustration of the glycan pool. In recent years, high performance liquid chromatography online coupled to mass spectrometry became very popular. By choosing porous graphitic carbon as a stationary phase for liquid chromatography, even non derivatized glycans can be analyzed. Detection is here done by mass spectrometry, but in instead of
MALDI-MS, electrospray ionisation (
ESI) is more frequently used.
Multiple Reaction Monitoring (MRM) Although MRM has been used extensively in metabolomics and proteomics, its high sensitivity and linear response over a wide dynamic range make it especially suited for glycan biomarker research and discovery. MRM is performed on a triple quadrupole (QqQ) instrument, which is set to detect a predetermined precursor ion in the first quadrupole, a fragmented in the collision quadrupole, and a predetermined fragment ion in the third quadrupole. It is a non-scanning technique, wherein each transition is detected individually and the detection of multiple transitions occurs concurrently in duty cycles. This technique is being used to characterize the immune glycome.
Table 1:Advantages and disadvantages of mass spectrometry in glycan analysis
Arrays Lectin and antibody arrays provide high-throughput screening of many samples containing glycans. This method uses either naturally occurring
lectins or artificial
monoclonal antibodies, where both are immobilized on a certain chip and incubated with a fluorescent glycoprotein sample. Glycan arrays, like that offered by the
Consortium for Functional Glycomics and Z Biotech LLC, contain carbohydrate compounds that can be screened with lectins or antibodies to define carbohydrate specificity and identify ligands.
Metabolic and covalent labeling of glycans Metabolic labeling of glycans can be used as a way to detect glycan structures. A well known strategy involves the use of
azide-labeled sugars which can be reacted using the
Staudinger ligation. This method has been used for in vitro and in vivo imaging of glycans.
Tools for glycoproteins X-ray crystallography and
nuclear magnetic resonance (NMR) spectroscopy for complete structural analysis of complex glycans is a difficult and complex field. However, the structure of the binding site of numerous
lectins,
enzymes and other carbohydrate-binding proteins has revealed a wide variety of the structural basis for glycome function. The purity of test samples have been obtained through
chromatography (
affinity chromatography etc.) and analytical
electrophoresis (
PAGE (polyacrylamide electrophoresis),
capillary electrophoresis,
affinity electrophoresis, etc.). ==See also==