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Capillary electrophoresis mass spectrometry for the characterization of glycoproteins and N-glycans

University of British Columbia

Glycosylation of proteins is ubiquitous and has the ability to significantly alter the biological and biophysical properties of proteins. The need to study structure-function relationships of glycans in a living organism requires the continuous development of rapid and sensitive technologies for the characterization of glycan components. In recent years, a broad range of technologies have evolved to provide new developments and emerging glycomics techniques. In this thesis we present the development of new methodologies based on capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) for the study of protein N-glycosylation in complex biological systems and therapeutic recombinant drugs. Our approach involves the use of a flow-through microvial, a novel technology that provides a robust and easy-to use strategy for interfacing CE separation with MS detection. In chapter 2, we report a simple and robust CE-ESI-MS methodology for comprehensive characterization of glycosylated proteins at the level of intact protein, enzymatically released glycopeptide and glycans. In chapter 3, we characterize a complex set of enzymatically released N-glycans from a recombinant therapeutic drug that revealed extensive glycan heterogeneity. The study demonstrated the potential of our approach to complement established techniques for glycan characterization of therapeutic glycoproteins in the pharmaceutical industry. Chapters 4 and 5 of this thesis are devoted study protein glycosylation of relevant biological systems. In chapter 4, O-acetylated N-glycans from fish serum were characterized in their native state and the structural variations of their isomeric species were investigated by tandem MS approaches. The developed CE-MS methods may be useful not only for the characterization of acetylation of complex glycans but also to study other types of glycan modifications in different contexts. In chapter 5, we present CE-MS methodologies for characterizing protein N-glycosylation in human serum associated with prostate cancer and asthma. Comparison of glycan compositions and relative abundances revealed abnormal glycosylation in prostate cancer and asthma serum. The capability of our CE-ESI-MS method to perform global glycan profiling of human serum demonstrates its potential for comprehensive glycan profiling in the context of malignancies and for the discovery of glycan disease markers with high selectivity and specificity.

Text

2015-12-31

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/51488

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/