Bottom up proteomics of cryptocyanin protein and anti-tumor Thomsen–Friedenreich neoglycoconjugate vaccine

Demian, Wael (2015) Bottom up proteomics of cryptocyanin protein and anti-tumor Thomsen–Friedenreich neoglycoconjugate vaccine. Masters thesis, Memorial University of Newfoundland.

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Abstract

In this thesis, two different kinds of complex biomolecules were examined: cryptocyanin protein of snow crab (Chionoecetes opilio) and carbohydrate-protein neoglycoconjugate vaccine using mass spectrometry (MS) and tandem mass spectrometry (MS/MS). The main objective of the first study was to determine structural changes in snow crab cryptocyanin protein. Accordingly, cryptocyanin was isolated and purified from molting and non molting snow crabs. The purified molting and non molting cryptocyanins were sequenced by using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) and collsion induced dissocation tandem mass spectrometry (CID-MS/MS). The structural differences between molting and non-molting cryptocyanin were reported using bottom-up proteomics. The molecular masses of the snow crab during molting and non-molting stages were found to be respectively 67.6 kDa and 68.1 kDa. In the second study, two anti-tumor Thomsen-Friedenreich neoglycoconjugate vaccines were prepared by the Michael addition reaction, and their molecular composition was confirmed by MALDI-TOF-MS analysis. De novo peptide sequencing using electrospray ionization in the positive ion mode mass spectrometry (ESI-MS, + ion mode) and low-energy CID-MS/MS of trypsin digests using a quadrupole-quadrupole-time of flight –hybrid tandem mass spectrometer (QqTOF-MS/MS). The CID-analysis allowed us to identify three glycation sites for the glycoconjugate prepared with a TF: protein ratio of 2:1, and 14 glycation sites for the glycoconjugate prepared with a TF: protein ratio of 8:1. It should be noted that this work represents the only published report for the determination of the exact poistions of the carbohydrate haptens on this unique C-S linked glycoconjugate. In addition, this investigation constitutes a versatile example for the identification of accurate quality control necessary in commercial production of glycoconjugate vaccines. Indeed, the glycopeptides isolated were fully characterized during this work and may well represent useful reference compounds that can be used in standardization analyses. The presented two applications of this research indicate that the cohesive and clever application of MS continue to grow due to its high sensitivity, and its ability to analyze high molecular mass biomolecules such as the two complex biomolecules. In summary this thesis has presented a major dynamic role for the identification, characterization and sequencing of two novel complex biomolecules namely the molting and non molting and cryptocyanin proteins and the two anti-tumor Thomsen-Friedenreich neoglycoconjugate vaccines. These biomolecules were chosen as elegant applications of MS/MS in the fields of proteomics and glycomics vaccine.

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