Author : Elisha Goonatilleke
Publisher :
Page : pages
File Size : 41,7 MB
Release : 2018
Category :
ISBN : 9780438930971
Book Description
Proteins are an important group of biomolecules known to provide a wide range of essential functions. Glycosylation, the enzymatic addition of sugar moieties to the protein, is a commonly occurring post-translational modification and adds greater diversity to the human proteome. Furthermore, protein glycosylation has been known to play key roles in all aspects of human development and health maintenance. Altered glycosylation in a physiological state has a detrimental effect on human health and even initiates many infectious and chronic diseases. However, structural heterogeneity, diversity in individual monosaccharides, and poor ionization efficiencies have made it more challenging to identify and characterize these glycans compared to the other biomolecules. This dissertation presents mass spectrometry-based methods developed to identify, characterize, and quantitate glycosylation in human proteins. Chapter I provides an overview of the structure and function of glycans and glycoproteins, along with key characteristics of mass spectrometry that are required for the analysis of those biomolecules. This includes liquid chromatography-based separation techniques, and mass spectrometry-based ionization and fragmentation methods. Chapter II presents a highly reproducible, sensitive, and rapid method to quantitate proteins and site-specific glycosylation in human milk across lactation using triple quadrupole mass spectrometry. Individual milk proteins including [alpha]-casein, [beta]-casein, [kappa]-casein, [alpha]-lactalbumin, lactoferrin, sectretory immunoglobulin A (SIgA), immunoglobulin M (IgM), immunoglobulin G (IgG) [alpha]-antitrypsin, osteopontin, and lysozyme carried a unique behavior across lactation while changes in site-specific glycosylation had an independent behavior than that of its protein. Chapter III presents a comprehensive comparative glycomic study of immunoglobulin A (IgA) in the milk, saliva, and plasma of individual donors. This study showed N-glycan-specific differences among three body-fluids and observed that the delivery mode can cause changes in IgA N-glycosylation in saliva and plasma IgA while milk IgA remained unchanged. Chapter IV shows a rapid method for the quantification of site-specific glycosylation and site-occupancy in recombinant monoclonal antibody drugs used in cancer therapeutics. Our results revealed that irrespective of the IgG subclass expressed in the drugs, the N-glycopeptide profiles are nearly the same but differ in abundances. Chapter V discusses an in-depth structural analysis of cell surface O-glycans using chip QTOF mass spectrometry.