Chemistry Reference
In-Depth Information
have one sialic acid residue in their non-reducing end, but on a different branch;
1A3-200.4 is sialylated on the
α
3Man branch, whereas 1A4-200.4 is so on the
α
6Man branch. Removal of the sialic acid produces the same product from both
isomers, and we can confi rm that both isomers have a sialic acid on the same
component, but cannot distinguish the branching pattern. The non- sialylated
branches can be digested by
-galactosidase. However, their elution positions still
remain very close to each other. Further treatment next with
β
-
N
- acetylhexosa-
minidase removes the GlcNAc residue and the resultant products have clearly
distinguishable elution positions. In this way, the estimated structure can be
ascertained through a combination of several degradation pathways.
β
5.5
Detailed Structural Analysis Using MS
Analysis using partial treatment is time consuming, and planning partial degrada-
tion requires detailed knowledge of enzyme substrate specifi cities and sample
specifi cities. MS analysis has also recently been applied for detailed structure
analysis [11-13]. As an example, negative-mode MS analysis with electrospray
ionization (ESI) of monosialyl-biantennary oligosaccharides shows a peak at
m
/
z
= 1003.9 (Figure 5.5a) [14]. This corresponds to the ionized PA-glycan after
removal of the two protons and charged
2, and is calculated as:
m
/
z
= 2010.9
(molecular weight of the PA- glycan) - 2 (molecular weight of the two protons
removed)/2 (number of charge). The four isomers described above show similar
spectra and cannot be distinguished. Tandem MS (MS/MS) is MS analysis in
which isolated peaks in the initial mass spectrum, in this case the ion at
m
/
z
= 1003.9, are broken by collision with helium gas. Each of the glycans shows
different fragmentation patterns (Figure 5.5b). As an example, the
−
α
3Man branch
cleaved at the Man
α
1,3Man linkage, whereas the
α
6 branch fragment included
Man
1,6Man. We can now, therefore, determine which branch is sialylated. The
linkage position of sialic acid is indicated by MS
3
analysis, because the
α
α
2,3 - linkage
is cleaved more easily than the
2,6-linkage. The ion of sialic acid alone is clearly
seen at
m
/
z
= 290 in the spectrum of the fragment from the glycan that includes
α
α
2,3 - sialylation (Figure 5.5 c).
5.6
Glycomic Analysis Using MS
MS is highly sensitive and rapid, and can be applied to glycomic analysis. This
approach to glycomics is used to determine biomarkers of disease. Sialic acid resi-
dues are often removed during MS analysis and the sensitivity of MS can vary
greatly depending on the charge of the sialic acid. Therefore, methylation (or
another method of capping the COOH on sialic acid) or permethylation (methyla-
tion of all OH groups on an oligosaccharide) is used in the MS analysis of sialyl
