Qualitative and Quantitative analysis of glycans and glycopeptides released from model glycoproteins and biological samples using MRM mode and ESI LC-MS/MS



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Glycosylation is one of the most important post translational modifications. Aberrant glycosylation has been correlated to a number of disease conditions. Changes in glycosylation can provide valuable information about the etiology of the disease state and also can be used as potential biomarkers for early prognosis and diagnosis. Currently, liquid chromatography interfaced to mass spectrometry is the most widely used techniques for the characterization of glycans and glycopeptides in order to detect changes in glycosylation. Development of reliable methods is required for correlation of these changes with the disease condition. The first part of the thesis describes method development and validation for the successful quantification of the glycopeptides using oxonium ions as transitions in multiple reaction monitoring (MRM) mode. The invariable presence of oxonium ions as a result of fragmentation of glycopeptides makes them an excellent choice as transitions for MRM of the glycopeptides. MRM on a triple quadrupole mass spectrometer enables very high sensitivity and is explored as a technique for quantification of glycopeptides. The various parameters influencing quantification of glycopeptides including MRM time segments, number of transitions and normalized collision energies were optimized. The results indicate that oxonium ions could be adopted for the characterization and quantification of glycopeptides in general, eliminating the need to select specific transitions for individual precursor ions. Also, enhanced sensitivity of analysis is attained when specific time segments are employed. This approach can also be applied to comparative and quantitative studies of glycopeptides in biological samples as illustrated for the case of depleted blood serum. The second part of the thesis describes characterization and quantification of glycans and glycopeptides of the aqueous humor samples collected from Fuchs endothelial corneal dystrophy (FECD) and normal subjects using LC-ESI-MS/MS. FECD is an eye disorder which results in progressive degeneration of the corneal epithelium causing impaired vision and in some cases total blindness. The exact biological events that cause this disorder are still unknown. In an attempt to establish a correlation between aberrant glycosylation and FCED, the glycome and the glycoproteome of aqueous humor was probed. The results suggest a significant difference between the disease and disease free states of one glycan. A general upregulation of glycan expression in the disease state was observed according to results of the glycomic study. These changes can be used to distinguish between the subjects with and without FECD. Also, these changes suggest that glycosylation is either an effect or cause of the disease condition.



Multiple reaction monitoring, Mass spectrometry, Liquid chromatography