Type of Document Dissertation Author Wang, xu URN etd-12122010-223757 Title Application of FT-ICR Mass Spectrometry in Study of Protein Modifications and Carbohydrates Degree Doctor of Philosophy Department Chemistry and Biochemistry, Department of Advisory Committee
Advisor Name Title Alan G. Marshall Committee Chair Qing-Xiang Amy Sang Committee Member Timothy M. Logan Committee Member Michael Blaber University Representative Keywords
- Mass Spectrometry
- Protein Posttranslational Modifications
- Bottom Up
- Top Down
Date of Defense 2010-11-23 Availability unrestricted AbstractProteins are organic compunds made of amino acids arranged in a linear chain and folded into a globular form. Protein synthesis follows the genetic code encoded on a sequence of gene. In the cell, shortly after protein biosynthesis, the amino acid residues in a protein are often chemically modified by posttranslational modification, which alters the physical and chemical properties, folding, stability, activity, and ultimately, the function of the proteins. The protein posttranslational modifications play a crucial role in generating the heterogeneity in proteins and also help in utilizing identical proteins for different cellular functions in different cell types. Also, the modifications play an important part in modifying the end product of protein expression and contribute towards biological processes and diseased conditions. Posttranslational modifications may happen in several ways, such as glycoslyaiton, phosphorylation, acetylation, methylation, lipoylation, etc. In addition, protein engineering may produce some unnatural modifications on the proteins for some specific research. My research interests mainly focus on study protein glycosylation and phosphorylation. During my graduate study, I have developed many methods to accomplish several projects.
Fourier transform ion cyclotron resonance mass spectrometer, FT-ICR MS, is particularly advantageous for protein modification analysis. High accurate mass collected from FT-ICR allows the reduction of precursor ion tolerances from the Dalton range to the order of few sub-parts per million, reducing the number of peptides initially considered by the peptide spectral matching algorithm. Chapter 1 introduces the theory of the instrument for mass measurement, ionization methods, MS-based strategies for protein modification analysis, and the biomolecule separation methods. Chapter 2 reviews phosphoproteomics and glycoproteomics by FT-ICR mass spectrometry. The contents of this review section cover: 1. background of phosphorylation and glycosylation; 2. sample preparation for mass spectrometry-enrichment and separation; 3. tandem mass methodology (CID, ECD, AI-ECD, ETD); 4. informatics.
Many applications have been applied to study protein modifications. Chapter 3 presents an application utilizing top-down and bottom-up proteomics with 14.5 T FT-ICR mass spectrometry to study sites and extent of selenomethionine incorporation into recombinant Cas6 protein. Cas6 is a novel endoribonuclease found in Pyrococcus furiosus. it cleaves CRISPR(clustered regularly interspaced short palindromic repeats) RNAs within the repeat sequences to release individual invader targeting RNAs. Selenomethionine modified proteins are commonly used to improve x-ray crystallographic structure resolution by multi-wavelength anomalous diffraction (MAD) phasing. However, the specificity and extent of selenomethionine incorporation must first be assessed before crystallization. Bottom-up and top-down proteomics with a modified 14.5 T LTQ FT-ICR MS offer a quick, accurate, and robust method to locate and quantify selenomethionine incorporation after auxotrophic expression. Comparative analysis confirmed that all six methionines were fully replaced by selenomethionines in Se-Cas6 (Wang X. et al., Rapid Commun. Mass Spectrom., (2010), 24 (16), 2386-2392).
A method applying LC ESI FT-ICR mass spectrometry for characterizing N-linked glycoproteins and assigning significant portion of glycopeptide along with composition/structural information of glycans is introduced in chapter 4. The method was first tested with a standard glycoprotein. Then it was applied to characterize N-linked glycoproteins in fetal bovine serum (FBS). N-linked glycans were enzymatically released from glycoproteins in FBS with PNGase F, followed by purification on a graphitized carbon cartridge SPE and separation on an TSK-Gel Amide80 column under hydrophilic interaction chromatography (HILIC) conditions. N-linked glycosylation sites were identified as well. To assign the glycopeptide compositions, experimentally determined glycan masses and glycosylation sites were applied. The masses of different combinations between glycan and peptide were searched in the mass measurements of glycopeptides. The uniquely identified masses were picked for assigning glycopeptides. In total, 137 unique glycopeptide compositions were assigned from 18 glycoproteins, and the glycan structures on most assigned glycopeptides were heterogeneous. High accurate mass measurements collected from FT-ICR MS provided confident identifications (Wang X. et al., Anal. Chem., (2010), 82 (15), 6542-6548).
Chapter 5, talks about characterization chemotherapies effect(s) of 2-deoxy-D-glucose (2-DG) on global glycosylation in glioblastoma derived cancer cells and cancer stem cells. 2-DG is a stable glucose analogue in which hydroxyl group at the second position carbon is replaced by a hydrogen. In our study, we found that the toxicity of 2-DG is mainly via interfering with N-glycosylation of proteins, disrupting protein foldings, leading to ER stress and further inducing apoptotic cell death. As results of 2-DG, inhibition of glycolysis and ATP depletion are not fatal for cell viability. The findings we are presenting clearly address the mechanisms of how 2-DG targets and kills cancer cells and cancer stems cells as a chemotherapeutic agents, which may be used for supporting further clinical trials of 2-DG. A conference presentation has been accepted by the 15th anual scientific meeting of the society for neuro-oncology, 2010, Montreal, Canada. The manuscript of these results will soon be submitted to Nat. Med.
Protein phosphorylation is a crucial event in most cellular processes. Since phosphorylation is always substoichiometric and the ionization efficiency of phosphopeptide is low during mass spectrometry analysis, selective enrichment will be required for large scale characterizing phosphorylation from complex biological samples. In chapter 6, an optimized phosphopeptide enrichment strategy consisted of calcium phosphate precipitation (CPP) coupling with TiO2 is addressed. The method was applied to study phosphorylations in androgen repressed cancer of the prostate (ARCaP) cells. Phosphopeptides in ARCaPs were analyzed on a hybrid LTQ FT-ICR mass spectrometer with LC MS/MS approach. After database search and stringent filtering, we generated a data set containing total of 385 high confident phosphoprotein identifications with overall mass error of 0.32 ± 0.6 ppm at peptide level.
Chapter 7 talks about that the phosphorylation/dephosphorylation regulates MSP fiber protein 3 (MFP3) in MSP-based amoeboid motility of Ascaris sperm. The crawling movement of nematode sperm requires coordination of leading edge protrusion with cell body retraction, both of which are powered by modulation of a cytoskeleton based on major sperm protein (MSP) ﬁlaments. We used a cell-free in vitro motility system in which both protrusion and retraction can be reconstituted, to identify two proteins involved in cell body retraction. Protein phosphatase 2A (PP2A), a protein unique to nematode sperm that binds to the MSP ﬁlaments in the motility apparatus, targeted MSP ﬁber protein 3 (MFP3). Dephosphorylation of MFP3 caused its release from the cytoskeleton and generated ﬁlament disassembly. Our results suggest that interaction between PP2A and MFP3 leads to local disassembly of the MSP cytoskeleton at the base of the lamellipod in sperm that in turn pulls the trailing cell body forward (Yi K., Wang X. et al., Mol. Biol. Cell, (2009), 20 (14), 3200-3208).
Chapter 8 presents comparative glycoproteomics data in a glioblastoma derived stem cell line, gCSC11, during differentiation. gCSC11 characterized by CD133 expression, grows as tumorsphere in culture and if subsequently implanted in nude mice brains, will recapitulate high grade glial tumors. Stimulation with a STAT3 inhibitor WP1193 or culturing in 10% FBS both led to loss of CD133 expression in gCSC11 cells, but differed in phenotype changes. STAT3 inhibitor treated gCSC11 cells underwent dissociation in culture and converted to progenitor cells if further treated with FBS. Glycoproteomics study revealed 33 differentially expressed glycoproteins, most of which has never been reported as glycosylated. ENO1 isoform alpha-enolase was preferentially expressed in native rather than in STAT3 or FBS treated gCSC11 cell. Preferential expression of alpha-enolase in gCSC11 glioblastoma stem cells might reflect the modulated metabolism or control the balance between self-renewal and differentiation via modulation of c-myc.
Chapter 9 talks about phosphorylation sites characterization of galectin-1 and its potential secretion/regulation pathway. Galectin-1 is a multi-functional protein performing either intracellular or extracellular functions as the monomer or homodimer, and it works on different cell types to put either positive or negative effects on cell growth. It is involved in tumor transformation, cell cycle regulation, apoptosis, cell adhesion, migration and inflammation. Galectin-1 is produced in the cytosol and can either remain intracellular or be delivered to the outside of cells via a non-classical secretion pathway. Galectin-1 lacks recognizable secretion signal sequences and does not pass through the standard ER/Golgi pathway. Thus, galectin-1 secretion must operate by a novel mechanism distinct from classical vesicle-mediated exocytosis. Our primary data of phosphorylation on galectin-1 may indicate the mechanism of its secretion/regulation pathway.
The appendixes include the papers I have published during my graduate study. Appendix A is the paper that covers the method development for characterizing N-linked glycans and glycopeptides by liquid chromatography electrospray ionization FT-ICR MS. Apendix B is a top-down mass spectrometry paper studying sites and extent of selenomethionine incorporation into recombinant Cas6. The publication in apendix C introduces a mechanism how MFP3 functions in cell body retraction, which was collaborated with Dr. Thomas M. Roberts in the biology department at Florida State University.
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