using fast atom bombardment (FAB) ionization and two coupled
analyzers (BE-EB). FAB ionization and the first analyzer (BE)
provided parent ions free from matrix contaminants, and structural
detail of these precursors was observed after collision and product
ion analysis in a second coupled analyzer (EB). Starting from
complex mixtures, this unique instrumental approach, BE-CID-
EB, contributed significantly to the GPI anchor structure obtained
from human acetylcholinesterase6 and the analysis of Rhizobial
signaling molecules in nitrogen fixation.7 These exciting structure-
functional studies heralded the prospects of gas-phase structural
analysis in the absence of chromatography, complete with the
sensitivity expected from electron multiplier detection. Develop-
ments, however, that have brought the most significant impact
to carbohydrate analysis were those leading to the dynamic
stabilization of ions in two- and three-dimensional radio frequency
quadrupole fields by Wolfgang Paul. These achievements led to
the subsequent development of the quadrupole mass spectrometer
and the ion trap8,9 and the 1989 Nobel Prize in physics. Thus,
with the ability to ionize samples at high mass and observe
fragments by CA in Paul ion traps, all the components were in
place for a full and detailed investigation of carbohydrate structure.
The first commercially available IT instruments were sold in 1984,
but it was another decade, when combined with ESI, that
carbohydrate analysis moved beyond the early success with triple
quadrupole instruments. First early reports demonstrated the
advantages of ITMS, but little attention was focused on defining
the limits of understanding carbohydrate structure.
Methylation. Derivatization has been a mainstay of oligosac-
charide structural investigations for over a century. Effective
analysis requires quantitative blocking of all hydroxyl groups, a
goal difficult to achieve, and that probably relates more to the
solubility of the reactants than the chemistry of the functional
groups. This aspect is rarely observed directly due to the extensive
organic extraction and washes with water that successfully remove
many polar undermethylated products. The first O-methyl ethers
of carbohydrate samples were prepared by Purdie and Irvine10
using methanolic solutions treated repeatedly with methyliodide
and silver oxide. Denham and Woodhouse11 and Haworth12
reported comparable results a few years later using dimethyl
sulfate and sodium hydroxide. The popular Purdie technique was
significantly improved by Kuhn and co-workers,13 who carried out
the reaction in the polar solvent N,N-dimethylformamide. All
methods, however, gave partially methylated products and ap-
proached completion only with repeated steps. Hakomori14 dem-
onstrated full methylation in one step using sodium methylsulfinyl
carbanion and methyl iodide dissolved in dimethyl sulfoxide
(DMSO). This method remained the procedure of choice for
another decade. The most recent improvement, and clearly the
simplest approach, has been that of Ciucanu and Kerek.15 To a
sample dissolved in DMSO, they added a slurry of sodium
hydroxide followed by methyl iodide. The overall simplicity and
the relatively low spectral background have now made this method
the most popular. Over the years, methylation has resolved a
number of major structural problems and along with acetylation
has dominated the Haworth-Birmingham carbohydrate school
for years. Haworth’s application of methylation provided a whole
range of crystalline well-characterized methyl ethers and their
oxidized products clarified Fischer ring structures, resolving the
20-year Haworth-Hudson controversy. This stemmed from the
assumption that the isomeric methyl glycosides discovered by
Emil Fischer in 1893 were to have five-membered, oxygen-
containing rings, a point disproved by Haworth and his associates
by methylation, which established a six-membered, oxygen-
containing ring. Hudson claimed the methylation strategy altered
the ring structure and the solution invalid and sought unsuccess-
fully to determine the true ring structure by correlation of
structure and optical rotation. More contemporary applications
were methylation analysis to characterize methylated alditol
acetates by GC/MS.16 Thus, mass spectrometry was combined
with methylation quite early, first to stabilize structures from the
more energetic ionization procedures, second for the advantages
provided by sample cleanup using lipophilic extraction, and finally
for the enhanced ion signals. Unraveling the details of structure
by CID and the advantages provided by methylation has been an
evolving process aided significantly by improved instrumentation.
Such combinations provided the structural details of a GnT-I
metabolite,17 profiling EPO Lys-C digests and their N- and O-linked
glycans structures,18 defining Rhizobial exopolysaccharide binding
epitope,19 lipid A metabolites,20 GSL structures,21-23 and unraveling
a unique bacterial endoglycosidase O-linked glycan structure.24
Many of these reports have been summarized.25,26
The simple principle of derivatizing all available hydroxyls
within an oligosaccharide and characterizing the products follow-
ing fragmentation has not changed, and we extend that principle
in this report using ITMS instrumentation for disassembly and
characterization.
Metal Ion Adduction. It is generally assumed that no single
analytical technique would be capable of a complete oligosaccha-
ride characterization. Even within a single instrumental approach,
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(16) Bjorndal, H.; Hellerquist, H. G.; Lindberg, B.; Svensson, S. Angew Chem.,
Int. Ed. Engl. 1970, 9, 610.
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1988, 263, 18776-18784.
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