Deciphering Branched Galactomannan Structures via Multistage Ion Mobility MS and MSⁿ Fragmentation (Multistage IMSⁿ)

Hemicelluloses are abundant carbohydrate components in plant cell walls. They play a crucial structural role in binding cellulose microfibrils, in addition to other biological functions. Their high structural variability directly influences their biological properties, making it important to establish a structure-function relationship. In the case of galactomannans, this complexity lies in branching, large size, in addition to isomerism, which makes their characterization challenging. In this context, we have demonstrated that cyclic ion mobility spectrometry (IMS), combined with porous graphitic carbon (PGC) chromatography, mass spectrometry (MS), and multistage MS/MS fragmentation (IMSⁿ), is a powerful tool for the detailed elucidation of galactomannan structures.

In this study, we show that our multistage IMSⁿ sequencing approach, previously validated for homo-linear oligosaccharides (OS), can be successfully applied to hetero-branched hemicelluloses with careful adjustments. Our approach consists of building a database (DB) library of high-resolution IMS (HR-IMS) profiles of disaccharidic and trisaccharidic fragments. Then, the sequence of the oligosaccharide of interest is retrieved by comparing the HR-IMS profile of its disaccharidic and trisaccharide fragments with the profiles from the DB library.

In fact, our IMSⁿ experiments on galactomannan reveal arrival time distribution (ATD) profiles matching with known reference structures, confirming the co-existence of multiple isomers. In addition, we proved that this approach could be improved by incorporating trisaccharidic fragments to our DB library, serving in the characterization of higher degree of polymerization (DP) structures (DP4 in this case).

Overall, this work paves the way for the characterization of even more complex oligosaccharides, which can be potentially used for bio-based material conception.