Analytical Aspects: Analysis of Protein - Bound Glycans - The Sugar Code - page 71

5
Analytical Aspects: Analysis of Protein-Bound Glycans
Hiroaki Nakagawa
The preceding chapters have introduced the biochemical basis and coding capacity
of the sugar code and given us an idea of the three-dimensional structure and the
chemical strategies for preparing glycans. Now we turn to analytical methods to
delineate glycan structures. As described in Chapter 1, the structural variability
poses a considerable challenge. For the analytical chemist the enormous coding
capacity means that not only are there a lot of possible structures (please see Table
1.1 for a telling example), but also that most samples include several kinds of
structures. In this chapter, analytical methods are described for detailed structural
analysis and glycomics (Figure 5.1 and Table 5.1). The processing results in the
complete structural description and glycomic profi ling of glycan mixtures.
5.1
Detection of Glycans on Glycoproteins
The presence of sugar chains attached to proteins can be detected using the peri-
odic acid-Schiff reaction (PAS) or lectin staining following sodium dodecylsul-
fate - polyacrylamide gel electrophoresis ( SDS - PAGE ). PAS staining was developed
by R. A. Kapitany and E. J. Zebrowski in 1973. It is based on the reaction between
an aldehyde, formed by the periodic acid, and Schiff's reagent. The original Schiff's
reagent (magenta) has been improved and several staining kits are now commer-
cially available. PAS is a general stain for carbohydrates and so is widely used in
histochemical studies. Lectin staining can distinguish distinct glycan structures
on glycoproteins. A list of lectin-reactive epitopes is given in Table 18.1. The
presence of an N -glycan can also be detected by shifts in molecular weight after
treatment with N -glycosidases, which remove the N - glycan from proteins. The
molecular weight changes can be detected using SDS- PAGE, high - performance
liquid chromatography (HPLC) or mass spectrometry ( MS ).
Amino sugars, N - acetylglucosamine ( GlcNAc ) and N - acetylgalactosamine
(GalNAc) can be demonstrated by amino acid composition analysis. The presence