High resolution mass spectrometry, especially Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS) is a widely practiced technique of choice in proteomics and lipidomics due to its high sensitivity, reproducibility and wide dynamic range. FT-ICR MS enables quick assignments of hundreds of peptides and lipids with extreme complexity. Chapter 1 introduces the fundamental of FT ICR phenomena for mass measurement and basic theories of LC-MS based hydrogen deuterium exchange (HDX) technique for high order structure studies. Chapter 1 also introduces the application of mass spectrometry in lipidomics including lipid classification and MS analysis. Chapter 2 described the characterization of the binding interfaces in R2TP complex by hydrogen/deuterium exchange mass spectrometry. The two closely related AAA+ family ATPase Rvb1 and Rvb2 form a tight functional complex with two Hsp90 interactors: Pih1p and Tah1p. The R2TP complex involves in multiple biological processes including apoptosis, PIKK signaling, and RNA polymerase II assembly, and snoRNP biogenesis. The current lack of structural information on R2TP complex prevents a mechanistic understanding of many biological processes. By use of solution-phase HDX MS, we probed the contact surfaces on Pih1p-Tah1p upon Rvb1/2p binding. The present results demonstrate that Pih1p-Tah1p interacts with Rvb1/2p through N-terminal and IDR2 regions of Pih1p. Significantly, HDX also detected a rearrangement of residues 38–60 of Pih1p and 1–44 of Tah1p upon formation of the R2TP complex. Chapter 3 depicts the study of conformations of activated, disease-associated glucokinase variants by a comparative hydrogen/deuterium exchange mass spectrometry. Human glucokinase (GCK) acts as the body’s primary glucose sensor and plays a critical role in glucose homeostatic maintenance. Previous biochemical and biophysical studies suggest the existence of two activated variants. HDX results demonstrate that a disordered active site, which is folds upon binding of glucose, is protected from exchange in α helix variant. Additionally, α helix variant displays an increased level of exchange near enzyme’s hinge region. In contrast, β hairpin variant does not show substantial difference in global or local exchange relative to that of wild type GCK. The work elucidates the structural and dynamics origins of GCK’s unique kinetic cooperativity. Chapter 4 investigated the structure of an antibody with ‘Knob-into-hole’ mutations by HDX MS. Bispecific antibodies (BsAbs) have flourished in the biopharmaceutical industry for targeting two distinct antigens simultaneously. ‘Knob-into-hole’ approach is a way to manufacture bispecific antibodies. The applicability and advantage of ‘Knob-into-hole’ engineered bispecific antibody is vast. However, concerns about the conformational change and immunogenicity risks posed by the new approach has have been raised. To better understand the conformations and dynamics impacted by the ‘knob’ and ‘hole’ mutations, HDX MS is used to characterize peptide-level conformational changes of a ‘Knob-into-hole’ engineered antibody. The study shows that there is no significant structural alternation induced by ‘Knob-into-hole’ framework. In Chapter 5, the applicability of resolving HDX-derived isotopic fine structure by ultrahigh resolving power FT ICR mass spectrometry was discussed. In an HDX experiment, labeling protein with deuterium causes the deuterium incorporation, resulting in distributions of various combinations of 13C1H and 12C2H (Δm = 2.9 mDa). The isotopic fine structure typically cannot be used to evaluate deuteration level due to the difficulty of .resolving fine structures for all proteolytic peptides spanning wide mass range from HDX experiments. The introduction of hexapolar cell triples the observed resolving power on 14.5 tesla FT-ICR mass spectrometer, thus we successfully extend the capability of resolving isotopic fine structure to most of identified peptides. Additionally, a new method of analysis of isotopic fine structure-resolved HDX data was proposed to determine degrees of deuterium incorporation. Another research area I have worked on is characterization of polar lipids by LC coupled with FT-ICR mass spectrometry. Algae lipids contain long-chain saturated and polyunsaturated fatty acids. The lipids may be transesterified to generate biodiesel fuel. In Chapter 6, I compared polar lipid compositions for two microalgae, Nannochloropsis oculata and Haematococcus pluvialis, that are prospective lipid-rich feedstock candidates for an emerging biodiesel industry. Online nano liquid chromatography coupled with negative electrospray ionization 14.5 T Fourier transform ion cyclotron resonance mass spectrometry ((−) ESI FT-ICR MS) with newly modified ion optics provides ultrahigh mass accuracy and resolving power to identify hundreds of unique elemental compositions. Assignments are confirmed by isotopic fine structure for a polar lipid extract. Collision-induced-dissociation (CID) MS/MS provides additional structural information. H. pluvialis exhibits more highly polyunsaturated lipids than does N. oculata. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2018. / June 13, 2018. / FT ICR Mass Spectrometry, Hydrogen Deuterium Exchange, Isotopic Fine Structure, Lipidomics, Liquid Chromatography, Protein High Order Structure / Includes bibliographical references. / Alan G. Marshall, Professor Directing Dissertation; Hengli Tang, University Representative; John G. Dorsey, Committee Member; Brian G. Miller, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_650724 |
| Contributors | Liu, Peilu (author), Marshall, Alan G., 1944- (professor directing dissertation), Tang, Hengli (university representative), Dorsey, John G. (committee member), Miller, Brian G (committee member), Florida State University (degree granting institution), College of Arts and Sciences (degree granting college), Department of Chemistry and Biochemistry (degree granting departmentdgg) |
| Publisher | Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text, doctoral thesis |
| Format | 1 online resource (119 pages), computer, application/pdf |
Page generated in 0.0036 seconds