Semester

Fall

Date of Graduation

2021

Document Type

Dissertation

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Chemistry

Committee Chair

Glen Jackson

Committee Member

Stephen Valentine

Committee Member

Justin Legleiter

Committee Member

Peng Li

Committee Member

Roberta Leonardi

Abstract

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 CID spectra. CTD provided a series of informative and unambiguous fragments corresponding to cross-ring cleavages from both the reducing end—such as 2,5Xn, 1,4Xn and 1,5Xn ions—and the non-reducing end, such as 0,2An, 1,5An ions. However, like CID, CTD also generates glycosidic bond cleavages to provide information about the monomeric sequence of each sulfated oligosaccharide The observed cross-ring fragment ions and glycosidic cleavages provide useful information on linkage patterns, sulfate group location and repeating unit composition that isn’t typically possible using CID-MS/MS.

In the final experimental effort, the combination of CTD-MS with UHPLC is presented for the analysis of a complex mixture of acidic and neutral human milk oligosaccharides (HMOs). Both branched and linear HMO structures were studied to determine the fragmentation behavior of He-CTD. This study focuses on the performance of the identification of the sialylation/fucosylation pattern, the differentiation of isomeric structures, and the characterization of the glycan sequences. He-CTD again provided a series of informative and unambiguous fragments, including 0,3A2 and 0,4A2 ions, which were helpful in the identification of linkage isomers present among sialyllacto-N-tetraoses. Unambiguous ions identified as 0,3A2 and 0,4A2 enabled differentiation of the α2,3- vs α2,6-linked sialic acid residues present among sialyllacto-N-tetraose HMOs. He-CTD can differentiate structural isomers for both sialyllacto-N-tetraoses and lacto-N-fucopentaoses by providing unique unambiguous cross ring cleavages of the type 0,2An, 0,2Xn and 1,5An for individual isomers. Our results highlight that He-CTD of acidic and neutral HMOs can be efficiently used with an online separation technique for the characterization of complex oligosaccharide mixtures while preserving the labile groups such as sialic and fucose residues and hence. The three applications faced different challenges for isomer discrimination, but CTD proved to fast enough and efficient enough to provide uniformly reliable data for these challenging and important biological applications.

Embargo Reason

Publication Pending

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