Jahouh, Farid (2012) Oligonucleotides sequencing and glycation sites determination of neoglycoconjugate vaccines using tandem mass spectrometry. Doctoral (PhD) thesis, Memorial University of Newfoundland.
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Mass spectrometry (MS) has turn out to be a fundamental technology which has produced tremendous strides in the biological sciences. In the last three decades, MS has witnessed convincing growth. Mass spectrometry has rapidly evolved to the forefront of analytical techniques; its ability to analyze proteins, deoxyribonucleic acid (DNA) and carbohydrate biomolecules has been a major driving force in the field of proteomics, genomics and glycomics. The level of performance that is achievable with MS today allows scientists to study various biomolecules in ways that were inconceivable 25 years ago. In this thesis, two different kinds of biomolecules will be covered: oligonucleotides and carbohydrate-protein neoglycoconjugates. -- First of all, the differentiation and sequencing of three constitutional isobaric 18-mer DNA oligomers, namely: GATTC A TAG CTACGAATC 1 , A ATTCG TAG CTACGAATC 2 , and A ATTCG TAC CTACGAAT G 3 were investigated using electrospray ionization (ESI) mass spectrometry with a hybrid QqTOF-MS/MS instrument. Our main objective was to differentiate and to sequence these oligonucleotides. The hypothesis was that the single scan ESI-QqTOF-MS analyses of the DNA oligomers will afford the same series of deprotonated molecular ions and that low-energy collision tandem mass spectrometric analyses (CID-MS/MS) will allow us to differentiate and to sequence the three DNA oligomers using the Mongo Oligo Software. Another objective was also to establish a mass fingerprint of these three oligonucleotides. This will aid to characterize the structure of covalently linked carcinogens to these three isobaric oligonucleotides. -- The second part of the thesis, which includes several chapters, is concerned with the use of MS for the localization of the glycation sites of neoglycoconjugate vaccine models formed by the covalent attachment of specific immunogenic carbohydrates obtained from different virulent bacteria to the protein carrier bovine serum albumin (BSA). This can be accomplished using a methodology based on squaric acid chemistry which was developed to conjugate carbohydrate antigens to lysine residues in proteins. Three different examples of hapten-BSA glycoconjugate vaccines were used: the Vibrio cholerae O1 antigenic specific hapten conjugated to the BSA, the tetrasaccharide side chain of the Bacillus anthracis exosporium conjugated to the BSA, and finally the model β-D-galactopyranosyl-(1→4)-β- D-glucopyranoside (β-lactoside) conjugated to the protein carrier BSA. Initially, the recording of the MALDI-TOF-MS of the different hapten-BSA glycoconjugate vaccines allowed us to determine the hapten-to-BSA ratios. The neoglycoconjugates were then digested and analyzed by MALDI-TOF/TOF-MS/MS and LC-ESI-QqTOF-MS/MS for the determination of the glycation sites. Our hypothesis was that the trypsin digestion will not allow a complete digestion of the protein, since the glycated lysines are not reactive with trypsin. Thus we had to use another protease, the GluC V8 which is known to digest proteins at the C-terminus of the aspartic acid and glutamic acid residues. We also suspected that the LC-ESI-QqTOF-MS/MS analysis was going to afford the identification of a higher number of glycation sites than the MALDI-TOF/TOF-MS/MS analyses.
|Item Type:||Thesis (Doctoral (PhD))|
|Additional Information:||Includes bibliographical references.|
|Department(s):||Science, Faculty of > Chemistry|
|Library of Congress Subject Heading:||Mass spectrometry; Oligonucleotides--Analysis; Nucleotide sequence; Glycosylation; Glycoconjugates--Analysis; Vaccines--Analysis;|
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