New approaches for quantitative glycomics using mass spectrometry
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The ability to quantitatively determine changes in glycan expression is an essential component of comparative glycomics. However, the quantitative analysis of N-glycans is often hindered by the lack of consistent analysis across laboratories due to the lack of quantitation strategies available. This study describes multiple improved relative quantitation approaches to facilitate studies of comparative glycomics while providing insight into their strengths and weaknesses, so that glycomic investigators can make an educated choice of the strategy that is best suited for their particular application. Chapter 1 is an introduction of glycomics and the application of mass spectrometry in this field. Also, it briefly describes the currently available techniques for quantitative glycomics as well as the challenges. Chapter 2 describes a novel method for relative quantitation of N-glycans by isotopic labeling using 18O-water during the enzymatic release process. The 2 Da mass shift for the labeled species can be differentiated from their unlabeled counterparts by using high resolution mass spectrometers, such as Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometers. Chapter 3 describes a Selected Reaction Monitoring (SRM) approach coupled with Hydrophilic Interaction Liquid Chromatography (HILIC) separation for relative quantitation of isomeric N-glycans with different sialic acid linkages. By using a novel superficially porous particle Penta-HILIC column, sialylated N-glycan isomers differing in α2-3 and α2-6 linkages can be resolved chromatographically. Thus, the relative quantitation of each sialic acid linkage isomer can be obtained from a straightforward LC-MS experiment. Chapter 4 describes the evaluation of an 15N-labeled monoclonal antibody (mAb) as an internal standard for quantitative glycomics. This 15N-labeled mAb reference is aimed at providing an internal standard on the glycoprotein level for quantitative glycomics, especially for the quality control of the Immunoglobulin G (IgG) pharmaceutical product batch to batch. Chapter 5 describes a targeted discovery glycomics strategy for global glycomics using scheduled Selected Reaction Monitoring (SRM) coupled with HILIC separation. This hypothesis driven glycomics approach utilizes the predictability of HILIC separation and the sensitivity and selectivity enhancement of SRM detection, which enables high through-put and consistent identification and quantification of diverse glycans expressed in different biological systems.