Linear Ion Trap MSⁿ 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

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 Lolium perenne. 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(n) method for the separation and detection of fructan isomers and present here evidence for specific MS(n) fragmentation patterns associated with β 1-2 (inulins) and β 2-6 (levans) fructans. LC-MS(n) analysis of (13)C labeled fructan oligomers produced by L. perenne 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.

LC-MS analysis reveals the presence of graminan- and neo-type fructans in wheat grains

“…Data were collected in centroid mode and analyzed with Xcalibur 3.0 software. Carbohydrates were identified by injection of commercial available standards and enzymatically synthesized fructan standards (Verspreet et al, 2014b) and by comparison of the MS 2 spectra with earlier published data (Harrison, 2012;Harrison et al, 2012). The nomenclature of Domon and Costello (1988) was used for the designation of the fructan fragments formed.…”

“…LC-MS combines the separating power of liquid chromatography and the structural characterization capacity of mass spectrometry. This allows the detection and characterization of individual fructan oligosaccharides in complex fructan mixtures (Harrison et al, 2012), such as found in wheat grain extracts. Wheat grain fructan structure is investigated in different grain tissues because grain tissues are often separated during wheat processing.…”

Liquid chromatography/mass spectrometry analysis of branched fructans produced in vitro with 13C‐labeled substrates

“…4(b)) comprises a X 2 ** fragment ion (m/z 395) corresponding to a cross-ring cleavage product containing two labeled fructose units that were β(2,6)-linked to the initial fructan structure. [17] Only 1&6,6-KP (m/z 839) and 1,6,6-KP (m/z 839) can form this fragment, but the absence of the 677 fragment indicates that this is the MS 2 spectrum of 1,6,6-KP. Further evidence for the identity of these structures was found in the HCD spectra.…”

“…2), as previously seen for inulin-and levan-type fructans. [17] Comparison of the MS 2 spectra of native 1&6-KT ( Fig. 2(a)) and [UL-13 C 6 fru] 1&6-KT ( Fig.…”

Design and Performance Evaluation of a Linear Ion Trap Mass Analyzer Featuring Half Round Rod Electrodes

“…These advantages lead to a significant improvement of analytical performance such as sensitivity and dynamic range. Owing to its good performance and capability of tandem mass spectrometry, the linear ion trap mass spectrometer has been commercialized and used in many fields [4][5][6][7].…”