Linear Ion Trap MS<sup>n</sup> of Enzymatically Synthesized 13C-Labeled Fructans Revealing
Differentiating Fragmentation Patterns of β (1-2) and β
(1-6) Fructans and Providing a Tool for Oligosaccharide Identification
in Complex Mixtures
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Abstract
Fructans are polymeric carbohydrates, which play important
roles as plant reserve carbohydrates and stress protectants, and are
beneficial for human health and animal production. Fructans are formed
by the addition of β-d-fructofuranosyl units to sucrose,
leading to very complex mixtures of 1-kestose based inulins, 6-kestose
linked levans, and 6G-kestose derived neoseries inulins and levans
in cool season grasses such as <i>Lolium perenne</i>. The
identification of isomeric fructan oligomers in chromatographic analysis
of crude plant extracts is often hampered by the lack of authentic
standards, and unambiguous peak assignment usually requires time-consuming
analyses of purified fructan oligomers. We have developed a LC-MS<sup>n</sup> method for the separation and detection of fructan isomers
and present here evidence for specific MS<sup>n</sup> fragmentation
patterns associated with β 1-2 (inulins) and β 2-6 (levans)
fructans. LC-MS<sup>n</sup> analysis of <sup>13</sup>C labeled fructan
oligomers produced by <i>L. perenne</i> fructosyltransferases
expressed in yeast has enabled us to account for the observed fragmentation
patterns in terms of preferential cleavage of the glycosidic bond
between O- and fructose C2 in both inulins and levans and to differentiate
reducing-end from nonreducing end cross ring cleavages in levans.
We propose that higher order MS fragmentation patterns can be used
to distinguish between the two major classes of fructan, i.e., inulins
and levans, without the need for authentic standards