Type of Document Dissertation Author Xiong, Ying Author's Email Address email@example.com URN etd-01252005-124636 Title Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometric Analysis of Biological Molecules: Oligoribonucleotide and Membrane Proteins Degree Doctor of Philosophy Department Molecular Biophysics, Institute of Advisory Committee
Advisor Name Title ALAN G. MARSHALL Committee Member JOHN G. DORSEY Committee Member LLOYD M. EPSTEIN Committee Member NANCY L. GREENBAUM Committee Member TIMOTHY M. LOGAN Committee Member Keywords
- Ion Cyclotron Resonance
- Fourier Transform
- Mass Spectrometry
- Isotopic Depletion
- Membrane-associated Proteins
- in Vitro Transcription
- Monoisotopic Relative Abundance
- Protein Identification and Characterization
- Electrospray Ionization
- Mycobacterium Tuberculosis
- Transmembrane Alpha Helix
Date of Defense 2005-01-20 Availability unrestricted AbstractFourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, with ultrahigh resolving power and ultrahigh mass accuracy, has been involved in many fields, in particular, environmental studies and biological sciences since its inception by Comisarow and Marshall in 1974. During the past decade, its applications were dramatically extended and remarkable progress has been made, especially in the field of biology, such as biopolymer sequencing, determination of biopolymer identities, interactions and their three-dimensional structures/conformations, drug screening and disease diagnosis, and so on. This dissertation focuses on biological applications of two types of biopolymers: oligoribonucleotide analysis (Chapter 2) and membrane protein identifications and characterizations (Chapters 3-6).
Chapter 1 briefly describes the instrumentations of FT-ICR mass spectrometry and its applications in analysis of proteins/peptides, nucleic acids and polysaccharides. Many modifications have been recently made to our 9.4 T FT-ICR mass spectrometer, such as tilted wires between the rods of the ion accumulation octopole and introduction of both CO2 laser beams (for infrared multiphoton dissociation (IRMPD)) and electron beams (for electron capture dissociation (ECD)) to the ICR cell. Progress, methods and large amount of examples of FT-ICR mass spectrometry in biological applications have been presented. It demonstrates that FT-ICR mass spectrometry has been involved a lot in biology in the past and will keep functional in this field in the future.
Chapter 2 introduces enhancement of mass determination of oligoribonucleotides by electrospray (ESI) FT-ICR mass spectrometry and 13C, 15N double depletion. The procedure for synthesis of a 32-mer by in vitro transcription with 13C, 15N doubly depleted rNTPs is described in detail. Methods for removal of salts are also included. Mass spectra reveal greatly enhanced-abundance of monoisotopic ions (by a factor of ~100) and a narrower isotopic distribution with higher signal-to-noise ratio. The abrupt
onset and high magnitude of the monoisotopic species promise to facilitate accurate mass measurement of RNA's. More importantly, it shall play a key role in analysis of nucleic acid-protein, nucleic acid-nucleic acid and nucleic acid-cofactor complexes in H/D exchange experiments.
Chapter 3 made some preparations for subsequent analysis of Mycobacterium tuberculosis H37Rv membrane proteins. Standard membrane proteins, M2 proton channel from influenza A virus and purified M. tuberculosis H37Rv major membrane protein Rv2031c have been used to optimize the conditions for membrane protein analysis by FT-ICR mass spectrometry. The different behaviors of M. tuberculosis peripheral and integral membrane proteins on two-dimensional gels led to the possibility by 2-D gel electrophoresis and FT-ICR mass spectrometry of identifying M. tuberculosis H37Rv peripheral membrane proteins, but not integral membrane proteins. The challenge still remains to characterize membrane proteins due to the difficulty in isolating intact membrane proteins by two-dimensional chromatography.
Chapter 4 presents identification of M. tuberculosis H37Rv peripheral membrane proteins by conventional two-dimensional gel electrophoresis and ESI FT-ICR mass spectrometry. Due to the limitations for hydrophobic proteins on 2-DE gels, M. tuberculosis H37Rv less hydrophobic membrane proteins were separated on a 2-DE gel in large scale. Strong and clear-cut protein spots were in-gel digested by trypsin and the extracted tryptic peptides were analyzed by ESI FT-ICR mass spectrometry. Subsequent tandem mass spectrometry aimed to obtain sequence information to increase confidence in protein identification. As a result, 29 distinct M. tuberculosis H37Rv proteins have been identified with significant MOWSE scores (p<0.05) and at least two tryptic peptides from each identified protein identification were further confirmed by Mascot sequence query based on tandem mass spectrometry. Unfortunately, none of them were reported as novel.
To access integral membrane proteins that are poorly resolved on 2-DE gels (or hardly enter 2-DE gels), M. tuberculosis H37Rv very hydrophobic proteins were separated, instead, by one-dimensional SDS PAGE gel electrophoresis and the extracted tryptic peptides from each gel band were analyzed by nanoLC LTQ tandem mass spectrometry. Chapter 5 concludes that 349 M. tuberculosis H37Rv proteins were identified in total with at least two tryptic peptide matches and MOWSE scores greater than 75. Complementary to previous studies, 84 novel M. tuberculosis H37Rv proteins were reported, of which 42 were integral membrane proteins with at least one predicted transmembrane alpha helix (excluding the possible signal sequence if any).
Chapter 6 extends analysis of M. tuberculosis H37Rv membrane proteins from identification to characterization. Two interesting post-translational modifications (PTMs), glycosylation and phosphorylation were chosen for study. Fluorescence-based staining of polyacrylamide gels by Pro-Q Emerald and Pro-Q Diamond, and enrichment of glycopeptides by lectin (concanvalin A (ConA)) affinity chromatography have been described. Fluorescent signals were observed on polyacrylamide gels, suggesting the existence of glycoproteins and phosphoproteins. Tryptic glycopeptides were accumulated by lectin (ConA) affinity chromatography and then analyzed by ESI FT-ICR mass spectrometry. However, no success has been led to localization of glycosylation. It is probably because that the glycoproteins are in extremely low abundance.
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