Development and Application of Electrospray Ionization Mass Spectrometry (ESI-MS) Methods for Measuring Carbohydrate-Active-Enzyme (CAZyme) Kinetics
Creators: Li, Zhixiong
Publisher: name: University of Alberta Library
Year: 2023

<p>This thesis focuses on the development of electrospray ionization mass spectrometry (ESI-MS)-based methods for measuring carbohydrate-active enzyme (CAZyme) kinetics and their applications for profiling CAZyme specificity, glycoprotein remodeling and disease diagnosis.<br>
Chapter 2 introduces CUPRA-ZYME, a versatile and quantitative ESI-MS assay for measuring the kinetic parameters of CAZyme reactions, monitoring pathways and profiling substrate specificities. The method employs the recently developed competitive universal proxy receptor assay (CUPRA), which is implemented in a time-resolved manner. Measurements of the hydrolysis kinetics of CUPRA substrates containing ganglioside oligosaccharides by the glycosyl hydrolase human neuraminidase 3 serve to validate the reliability of CUPRA-ZYME for quantifying kinetic parameters and highlight its use in establishing catalytic pathways. Application to libraries of substrates demonstrate the potential of CUPRA-ZYME for quantitative profiling of the substrate specificities of glycosidases and glycosyltransferases.<br>
Measuring CAZyme kinetics for glycoprotein substrates is challenging due to their heterogeneity. Chapter 3 introduces a simple but quantitative ESI-MS method suitable for glycoprotein substrates. The assay, referred to as Center-of-Mass (CoM) Monitoring (CoMMon), relies on continuous monitoring of the CoM of an ensemble of glycoprotein substrates and their corresponding CAZyme products. Notably, there is no requirement for calibration curves, internal standards, labelling, or mass spectrum deconvolution. The reliability of the CoMMon method was demonstrated by neuraminidase-catalyzed cleavage of N-acetylneuraminic acid (Neu5Ac) residues from a series of glycoproteins of varying molecular weights and degrees of glycosylation. Reaction progress curves and initial rates determined by CoMMon are in good agreement with results obtained, simultaneously, using an isotopically-labeled Neu5Ac internal standard, which enabled<br>
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the time-dependent concentration of released Neu5Ac to be precisely measured. To illustrate the applicability of CoMMon to glycosyltransferase reactions, the assay is used to measure the kinetics of sialylating a series of asialoglycoproteins by the human sialyltransferase ST6Gal1. The kinetic data show no correlation between initial rate and the number of acceptor sites. Moreover, the apparent kinetics are well described by double exponential functions. This finding, combined with the results of high-performance liquid chromatography (HPLC) analysis of the N-glycans released from the substrates during the sialylation reaction, suggest that ST6Gal1 has a preference for the α1-3 branch.<br>
Drawing on the methodological advances made in Chapter 2 and 3, a top down MS method for quantifying the α2-3- and α2-6-linked Neu5Ac content of prostate specific antigen (PSA) is introduced in Chapter 4. The assay employs CoMMon and a combination of specific (for α2-3-linked Neu5Ac) and nonspecific neuraminidases. Moreover, it is free of errors associated with lectin-based quantification of N-glycans containing both α2-3- and α2-6-linked Neu5Ac and avoids the sample handling steps required for HPLC analysis. The assay is validated using PSA from a commercial source and PSA standards containing all α2-3- or α2-6-linked Neu5Ac. The α2-3 Neu5Ac content of a PSA standard measured with the assay agrees with values obtained by HPLC analysis of released N-glycans. To illustrate the potential of the assay for clinical diagnosis of prostate cacer (PCa) and disease staging, the relative α2-3 Neu5Ac content on PSA extracted from serum of low, intermediate and high risk PCa individuals are determined. The results indicate a high sensitivity and specificity for discrimination of both low risk PCa and high risk PCa or low and intermediate risk PCa.</p>