A workflow for comprehensive structural determination of fructans and raffinose family oligosaccharides

Fructans (FOS) and raffinose family oligosaccharides (RFOs) are two major water-soluble carbohydrates in higher plants. Despite the development of diverse analytical methods, determining the complete FOS and RFOs structures remains challenging. A workflow that can be directly applied for comprehensive structural determination of FOS and RFOs was developed in this study.

The first step of the workflow follows a conventional strategy: HPLC retention times and MS² spectra of unknown oligosaccharide are compared to those of standards in a database. Structures that can be determined in this step are limited to the available standards. For oligosaccharides that do not match database entries, the second step is applied. Individual isomers are subjected to acid and/or enzyme hydrolysis to generate disaccharides and smaller oligosaccharides. Structures of these disaccharides/small oligosaccharides are determined by comparing HPLC retention times and MS² spectra with database. Complete oligosaccharide structure is reconstructed from these identified disaccharides/small oligosaccharides using logically derived sequence tandem mass spectrometry (LODES/MSⁿ). In the third step, new disaccharides and small oligosaccharides generated by acid/enzyme hydrolysis and the new oligosaccharides which structures are determined in the second part are added into database. The new data benefits the next oligosaccharide structural determination.

As this workflow is repeatedly applied to find new oligosaccharides, the database may eventually cover most natural FOS and RFOs. We first applied workflow to FOS extracted from onion and identified a previously unreported tetrasaccharide in onion, then applied to other species, demonstrating the capability for rapid isomeric identification of FOS and RFOs.