268
Carbohydrate Analysis
residues. These have free hydroxyl groups
in the positions where they were linked
to each other and methyl ether groups
where the original carbohydrate had free
hydroxyl groups. The products are further
derivatized to render them volatile and are
analyzed qualitatively and quantitatively by
GC coupled to electron impact ionization
spectroscopy (EI-MS). This MS produces
very energetic ions that fragment read-
ily, nearly always resulting in the loss of
an intact molecular ion. It is the break-
down pattern rather than the molecular ion
that enables us to distinguish between the
methylated carbohydrate residues (Fig. 8).
The positions of the acetyl groups indi-
cate where the unmethylated free hydroxyl
groups were in the partially methylated hy-
drolyzed sugars, thus showing where the
residue was substituted. However, it gives
no information on either the sequence of
these residues or their anomeric confor-
mation.
Naturally methylated or acetylated gly-
cans may be analyzed by methylation,
as just described, but using deuterated
methyl iodide or acetic anhydride to distin-
guish between the natural and chemically
derivatized sites by their different masses.
2.4.4
Mass Spectrometry
Mass spectrometry is capable of provid-
ing sequence information in addition to
its previously described use, determining
molecular weight. In FAB-MS, as little as
1pmo
lo
fg
l
y
c
ans
amp
l
ei
sbomb
a
rd
ed
with an accelerated beam of neutral atoms
(
e
.
g
.X
e
,C
s
)inav
a
cuum
.Th
i
sc
au
s
e
s
desorption of the sample from the liquid
surface, forming a characteristic molecular
ion plus a wealth of structurally diagnostic
fragment ions derived from it. The struc-
ture of a sample is determined given the
known fragmentation patterns of related
model structures. Improved spectra can
usually be obtained by the use of fully
methylated or acetylated carbohydrates.
As fragmentation predominantly occurs
between glycosidic links, the breakdown
pattern may give an abundance of infor-
mation concerning the glycan’s sequence.
As an example, the biantennary glycan
(Fig. 2b) when fully methylated gives the
following
m
/
z
peaks:
2492
(Neu5Ac
Gal
GlcNAc
Man)
2
Man
GlcNAc)
+
1029
(Neu5Ac
Gal
GlcNAc
Man)
+
825
(Neu5Ac
Gal
GlcNAc)
+
376
(Neu5Ac)
+
Although linkage information is pro-
duced, the MS is not able, in this case,
to differentiate the order of the hexoses
or
N
-acetylhexosamines, nor is it able to
determine the linkage positions.
Tandem mass spectrometry (MS/MS)
utilizes two or more mass-selection de-
vices in series so that the known ion from
one analyzer may be selected and induced
to give daughter fragments that are deter-
mined using a second analyzer. It can
provide complete sequence, branching,
and linkage information. Even pyranose
and furanose ring systems may be dis-
tinguished owing to differences in their
cleavage patterns. Tandem mass spectrom-
etry can be used with FAB, ESI, or MALDI
and the use of post source decay (PSD) in
MALDI-TOF or collision-induced dissoci-
ation (CID) in ESI has proved particularly
useful as the extent and distribution pat-
tern of fragments is highly reproducible.
2.4.5
NMR Spectroscopy
The power of NMR is most useful when
it is used to determine the sequence,
linkage positions, and conformation of
carbohydrates.
Linkage
and
sequence
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