Analysis of Saponin Mixtures from Alfalfa ( Medicago Sativa L. ) Roots using Mass Spectrometry with MALDI Techniques

Abstract

This model study demonstrates the value of Matrix Assisted Laser Desorption Ionization (MALDI) MS in global analysis of mixtures of saponin isolates. Unfractionated saponin extract derived from alfalfa [Medicago sativa L.] roots was analyzed. In addition, a few saponin fractions that were purified from the same extract by a series of chromatographic steps were also studied. MALDI mass spectrometry utilized TOF, TOF/TOF and QqTOF analyzers. Low-resolution fingerprints of the mixture characterized the sample in terms of a minimal number of distinct saponin components. The main species observed under MALDI-TOF and oMaldi-QqTOF MS positive ion mode conditions were sodiated pseudomolecular ions [M+Na]⁺. No protonated molecular ions [M+H]⁺ were detected. The MS/MS spectra acquired on [M+Na]⁺ precursor ions under conditions of collision induced dissociation (CID) were dominated by cleavages at glycosidic bonds. Product ions representing free aglycones were either absent or present at low intensities and were never observed for structures carrying an oligosaccharide bound to a sapogenin at the C3-O-position. In general, product ion series generated from CID fragmentation of glycans bound via an ether bond (e.g., C3-O-position in medicagenic acid) were consistent with the gas phase cleavages of each of the glycosidic bonds within the oligosaccharide, thus revealing the primary structure. In contrast, glycans bound via an ester bond (e.g., C28-O-position in MA) were released as intact sodiated species. A total of 78 pseudomolecular ions demonstrating signal-to-noise ratios above 5 were observed in the MALDI-TOF mass spectrum of unfractionated root extract from M. sativa. Molecular masses of 52 out of 78 were consistent with at least one known or novel saponin structure, with 10 of those 52 likely representing doubly sodiated saponin species. Calculated masses of the majority of the known M. sativa saponin structures were matched to experimental pseudomolecular ion masses. MS/MS analysis of unfractionated extract allowed us to propose putative structures for 51 saponins: 15 of these corresponded to the known M. sativa species, 8 to other Medicago genus species and 26 were not reported before for Medicago genus. We submit that the approach described here might serve as a high throughput strategy for evaluating effects of stressors or genetic manipulation on the overall composition of the saponin content of an organism.