Online Ultra-High Performance Liquid Chromatography-Charge Transfer Dissociation-Mass Spectrometry (UHPLC-CTD-MS) of Complex Mixtures of Oligosaccharides

Abstract

Online Ultra-High Performance Liquid Chromatography-Charge Transfer Dissociation-Mass Spectrometry (UHPLC-CTD-MS) of Complex Mixtures of Oligosaccharides Praneeth M. Mendis Complex carbohydrates make up more than half the biomass on earth, and they are crucial to a wide range of processes in living organisms. When polysaccharides and glycans are enzymatically digested into manageable units called oligosaccharides, they typically consist of 2-20 linear or branched sugar units. The detailed structural analysis of oligosaccharides assists in the identification of structural and chemical properties of the polymers from which they derive. Tandem mass spectrometry (MS/MS) is a key technique used in oligosaccharides structural characterization. Within tandem mass spectrometry, charge transfer dissociation (CTD) is a novel ion activation method that until now, has shown significant potential for the analysis of directly-injected oligosaccharide samples. In this dissertation, charge transfer dissociation was coupled with ultra-high performance liquid chromatography (UHPLC) for the first time to characterize complex mixtures of bioactive oligosaccharides. Three different examples are presented to test different performance capabilities relative to UHPLC with conventional collision-induced dissociation (CID). In first demonstration of online coupling, UHPLC-CTD-MS was applied to a complex mixture of highly methylated citrus pectin. Optimization studies included the solvent composition and gradient elution, ion source conditions—including voltages, drying gas flow rate, nebulizing gas flow rate and temperature—and CTD-MS conditions, like kinetic energy, flux and duration. CTD-MS acquisition rates were faster than 2 Hz, which demonstrates that spectral acquisition rates are fast enough to enable their coupling with UHPLC. Results from UHPLC-CTD-MS were compared to results obtained using UHPLC-CID-MS on the same instrument, and the CTD spectra contained more cross-ring fragments and fewer neutral losses, both of which assisted the structural characterization of the oligosaccharide mixture. UHPLC-CTD-MS successfully elucidated the structures of oligogalacturonan isomers, which were chromatographically resolved using ion-paired reversed-phase (IP-RP)-UHPLC. In the second study, the same UHPLC-CTD-MS system was adapted for the analysis of a mixture of sulfated oligosaccharides. The mixture contained kappa (κ), iota (ι), and lambda (λ) carrageenans that contain different degrees of sulfation ranging from one to three per repeating dimer, different positioning of the sulfate groups along the backbone, and varying degrees of polymerization (DP). Our results demonstrate that He-CTD is compatible with UHPLC conditions without any compromise in the amount of samples injected. Despite the lability of the fragile sulfate groups, most of the CTD fragment ions retained the labile sulfate groups whereas sulfate losses were abundant in the comparison