Type of Document	Dissertation
Author	He, Huan
URN	etd-03292009-200841
Title	Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry in Biological Applications: Lipids and Proteins
Degree	Doctor of Philosophy
Department	Chemistry and Biochemistry, Department of
Advisory Committee	Advisor Name Title Alan G. Marshall Committee Chair Andre Striegel Committee Member Mark R. Emmett Committee Member Michael Roper Committee Member Qingxiang Amy Sang Committee Member Hengli Tang Outside Committee Member
Keywords	Sulfatides Phospholipids Glycolipids Nano-LC Lipids FT-ICR MS Glycoproteins Cancer Stem Cells
Date of Defense	2009-03-18
Availability	unrestricted
Abstract Contrary to the tremendous amount of information on genomes and proteomes, coverage on lipidomes is limited. Polar lipids, for example, glycolipids and phospholipids, play important roles in cellular functions and not just provide structural support. Correlation of lipidome modulation with genome and proteome will help us better understand the underlying biochemical pathways in the disease states. High mass accuracy and high mass resolving power provided by FT-ICR MS combined with quick fragmentation techniques applied in the lower resolution LTQ MS enable us to quickly assign hundreds of polar lipid species from the cell cultures. Polar lipidomics study may lead to discovery of novel therapeutic targets/agents. Chapter 1 provides introductory information on FT-ICR MS basic theories, instrumentation features, application of liquid chromatography, mechanism of electrospray ionization, various fragmentation techniques, and finally lipid classification and MS analysis. Chapter 2 describes various aspects of a developed method for polar lipid semi-quantification. Simple liquid-liquid extraction and one-step nano-LC separation are important for analyses of complex cellular lipid mixtures – We observed more than 20 fold increases in detected lipid species after nano-LC separation. This method was applied on glioma cell lines, U87, under different combination of gene-/chemotherapy. Combination of adenovirus wt-p53 transfection and topoisomerase inhibition results in cells’ G2 arrest and apoptosis. A sugar binding protein, galectin-1, was down-regulated under such treatment. Lipidomics study identified the associated changes in the polar lipidome - the decrease of short chain asialo-GM1 (a ligand of galectin-1), decreases of long chain gangliosides, increases of short chain sulfatides and increases of all phospholipids, especially hydroxylated phospholipids. Chapter 3 characterized the modulation of cellular glycosphingolipids in NS11, a cancer stem cell line, under different differentiation pathways. Serum induced differentiation results in increase of glycosphingolipids. Such increase correlates with the increase of a glycogene responsible for the synthesis of a common precursor. STAT3 phosphorylation inhibited cancer stem cells showed decrease of glycosphingolipids and such decrease correlates with increase of a sialidase gene. Compared to differentiated cells, cancer stem cells displayed higher ratio of long chain/short chain glycosphingolipids, higher ratio of saturated/unsaturated long chain glycosphingolipids and lower level of sphingomyelins. In Chapter 4, cellular glycosphingolipids profiles were extensively studied in sialyltransferase overexpressed cell lines. Unexpected lack of GD1á and increase of GM2á suggest interesting cross-species substrate differences of ST6GalNAcV in human vs in mice. A true relative quantification method of cellular sphingolipids by metabolic labeling with light, heavy serine and serine synthesis inhibitor was described in Chapter 5. Heavy isotope incorporation was demonstrated and an interesting serine recycling pathway was identified. Characteristic isotope distribution also helped us to identify a class of sulfated glysphingolipids. A mass defect based glycopeptides search method was described in Chapter 6. Different from other reported methods for identification of glycopeptides, this method does not solely depend on the quality of provided protein sequences. This method was applied to identify 70 microheterogeneous glycopeptides in sRAGE protein, a therapeutic target for Alzheimer’s disease. In the course of my dissertation research, I enjoyed exposing to new developments of FT-ICR MS. Two published/accepted papers on electron capture dissociation (ECD) to differentiate between c and z ions (for protein de novo sequencing) and predict peptide gas phase secondary structure were listed in Appendices B and C. Another published paper on characterization of a novel crab peptide was listed in Appendix D. Finally, the published lipidomics methodology paper was listed in Appendix E.
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