Chemistry Reference
In-Depth Information
Table 5.1
Equipment and technologies in each step of the experiments described in this
chapter.
Step
Equipment and techniques
Sample preparation
detect glycan
PAS, lectin and other glycostaining methods with SDS-PAGE
N
- glycosidase treatment
amino acid analysis
release glycan
N
- glycosidase (
N
- glycan)
hydrazinolysis (
N
- glycan,
O
- glycan)
alkali degradation (
O
- glycan)
purifi cation
column chromatography or cartridge extraction (gel fi ltration,
ion exchange, reversed phase, cellulose, active carbon, lectins)
sugar - specifi c binding materials (hydrazide or oxime deriva-
tives)
derivatization
amine, hydrazine and oxime derivatives
methylation
Structural analysis
detailed structure
elution positions on HPLC
sensitivity for glycosidases
fragmentation on MS
glycomics
MS
other methods
CE, PAGE, HPLC
NMR, methylation analysis, lectin specifi city
glycopeptides
MS
of an
O
-linked glycan is likely if GalNAc is detected, since an
N
- glycan rarely con-
tains GalNAc (for an example, please see Figure 1.7b). In contrast, the detection
of mannose by monosaccharide composition analysis indicates the presence of an
N
-glycan, with the exception of
O
- mannosylation (see Chapters 7.3.3 and 22.2.2 ).
To make the strategy of this procedure clear, examples of detailed structural analy-
sis are instructive (see below).
5.2
Release of Glycans from Glycoproteins
N
-Glycosidase is eminently useful for examining oligosaccharides (see Info Box )
and it is also commercially available.
N
- Glycosidase
F
from
Flavobacterium
spp. does not cleave
N
-glycans that possess core
1,3 - fucose residues, known to
exist in plant and insect glycoproteins (please see Figures 8.3 a and 8.4 a) [1] .
N
-
Glycosidase A from almonds, however, does cleave these structures and is used
commonly.
N
-Glycosidase F is smaller than A and is sometimes used for natural
α
