Glycan Labeling and Analysis

In contrast to proteins and peptides, glycans do not absorb ultraviolet (UV) light strongly, thereby giving a weak detector signal even at 214 nm. Furthermore, as glycans with various different structures may be present in minute amounts in glycoprotein hydrolysates, their detection by UV absorbance may not be practical. As a result a wide range of alternative techniques have been developed for detection of glycans.

Instead of the UV detectors typically used for on-line HPLC systems, refractive index and pulsed amperometric detectors, with sensitivity in the range of 10 to 100 pmol, have enabled improved detection of native mono- and oligosaccharides. Another approach to improve glycan detection is radioisotopic labeling, but this technique is not widely used due to the safety and regulatory requirements for the handling of radiolabeled substances.

Most glycoproteins exist as a heterogeneous population of glycoforms, glycosylated variants with a single protein backbone and a heterogeneous population of glycans at each glycosylation site. It has been reported that for some glycoproteins, 100 or more glycoforms exist at each glycosylation site. In view of this heterogeneity and the presence of branched structures, the analysis of glycans is much more complicated than protein chemistry. It requires several different strategies to separate and study the structure of each individual glycan.

Once the glycans have been released from the glycoprotein, the glycan pool can be analyzed by MALDI-TOF (matrix assisted laser desorption/ionization time-of-flight) mass spectrometry or, after fluorescent labeling, by either HPLC or MS, or both. This strategy can provide a ′glycan profile′ or a ′glycosylation pattern′ that is highly characteristic of the glycoprotein. The technology can be applied to compare glycan profiles of glycoproteins found in normal and diseased states or to compare different batches of recombinant protein products. Chromatography combined with mass spectrometry provides valuable information in terms of potential composition. By using various specific exoglycosidases, structural information regarding monosaccharide identity and linkage orientation can be elucidated.

For more information on Glycan Labeling and Analysis please browse our online Glycobiology Analysis Manual.