262
Carbohydrate Analysis
600
800
1,000
1,200
1,400
1,600
1,800
2,000
m
/
z
80
60
40
20
100
TetraNA
3
4
+
TetraNA
4
4
+
TetraNA
4
3
+
TetraNA
3
3
+
TetraNA
3
2
+
TetraLacNA
4
4
+
TetraLacNA
4
3
+
Fig. 5
ES-MS of oxidized, reduced (NaBD
4
),
and methylated glycan from recombinant
erythropoietin. ES-MS produces multiply
charged ions. This example is centered on triply
positively charged ions. Periodate oxidation,
which oxidizes
cis
-diols only, of the glycans was
used to resolve isomeric structures and discover
linkage positions. For example, the most
abundant peak (TetraNA
4
,
m
/
z
1422.4, mass
4267.2 Da) is derived from a tetraantennary
glycan containing four terminal neuraminic acid
residues and a fucosyl residue (4633.5 Da). The
other major species, Tetra NA
3
and
TetraLacNA
4
, are derived from similar glycans
but with an
N
-acetylneuraminic acid residue loss
and an
N
-acetyllactosamine residue gain
respectively. Redrawn from Linsley et al. (1994)
with permission from Elsevier Science.
due to the absence of Frst a hexose and
then an
N
-acetylhexosamine residue. ES-
MS is ideally suited for direct interfacing
to HPLC and capillary electrophoresis. It
is far less able to handle buffers and deter-
gents than MALDI-MS, however.
Matrix-assisted laser-desorption ioniza-
tion mass spectrometry (MALDI-MS) gives
results similar to those obtained from ES-
MS but can extend the range of detectable
molecular ions. In this method, the ion-
ization of dry samples is achieved through
the use of a short pulse of intense UV-laser
light in the presence of a 100-fold excess of
a UV-absorbing organic molecule (known
as the matrix, e.g. 2,5-dihydroxybenzoic
acid). Even very large molecules can be
vaporized and ionized as MALDI is capable
of determining molecular weights, in ex-
ceptional cases, of up to
m
/
z
300 kDa using
time-of-flight (TO±) detection. It requires
only femtomoles to picomoles of mate-
rial, no sample derivation, and tolerates
more buffer and detergent than other MS
methods. Although able to handle pro-
teins, it has not had similar success, so
far, with natural polysaccharides in the
upper part of this range (
>
25 kDa) and
seems to work best with oligosaccharides
below 5 kDa. This may be partly due to the
polydispersity of such samples and partly
due to the inherently poor resolution of
MALDI-TO± MS.
Mass spectrometry must generally be
combined with other analytical methods of
analysis as it cannot distinguish between
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