Mass spectrometry methods for the structural analysis of glucan- and arabinoxylan-derived oligosaccharides

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Oligosaccharides potentially present in cereal matrices have attracted attention because of their health-related properties, such as prebiotic potential. In addition to those that are naturally present, oligosaccharides can be formed during processing by the hydrolysis of cereal polysaccharides, such as arabinoxylan, by endogenous or microbial enzymes. In sourdough fermentation, additional poly- and oligosaccharides may be produced by lactic acid bacteria (LAB). In the work performed for this thesis, mass spectrometry (MS) methods were implemented in the structural analysis of various enzymatically produced oligosaccharides. The research was focused on finding solutions to separating and identifying oligosaccharide isomers. To determine the linkage positions of linear mixed-linked glucooligosaccharides (GLOS), the feasibility of the positive and negative electrospray ionisation multiple-stage MS (ESI-ITMSn) were compared. Next, the ability of ESI-ITMSn to detect the branching points and linkage positions of complex arabinoxylan-oligosaccharides (A)XOS was studied. The MS method was also coupled to hydrophilic interaction liquid chromatography (HILIC) to separate and identify the (A)XOS in mixtures. Finally, the potential of travelling wave ion mobility spectrometry (TWIMS) combined with MS to differentiate the isomeric (A)XOS was evaluated. To study the structures of the isomeric acceptor products formed by dextransucrase from LAB, 13C-labelling was applied with ESI-ITMSn. The ESI-ITMSn method provided structural information about the molecular weights, sequences and linkage positions of the linear mixed-linked GLOS and (A)XOS. Negative ionisation was found to be more informative than the positive ionisation mode in analysing the linkage positions. The stepwise C-ion fragmentation in negative mode from the reducing end towards the non-reducing end allowed to study the linkage diagnostic fragment ions from the middle and the non-reducing end in both linear and branched oligosaccharides. O-2 or/and O-3 arabinofuranosyl substituents of AXOS were determined by the presence or absence of diagnostic ions in the ESI-ITMS3 analysis in the negative mode. The ESI-ITMSn method, combined with 13C-isotope labelling, enabled the analysis of unique trisaccharides produced by dextransucrase as well as the further detailed analysis of the isomeric fragment ions. Coupling tandem mass spectrometry (MS/MS) to HILIC enabled the separation and identification of all AXOS by the combination of retention times and the MS/MS spectra. CID-TWIMS-MS/MS was shown to be a powerful tool in differentiating the isomeric AXOS when both the fragment ions and the precursor ions were analysed.

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