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MASS SPECTROMETRY DISSOCIATION STUDIES OF PROTEIN-PROTEIN AND PROTEIN-NUCLEIC ACID COMPLEXES AND 13C FLUX OF AMINO ACIDS

Ma, Xin
http://rave.ohiolink.edu/etdc/view?acc_num=osu1397773864

Abstract Details

2014, Doctor of Philosophy, Ohio State University, Chemistry.
The development of analytical techniques for biological molecules provides analytical tools for biochemistry and structural biology, and the application of mass spectrometry (MS) in this area is growing fast. This dissertation illustrates the applications of MS in biological analysis from monitoring amino acids in metabolism to determining the quaternary structure and stoichiometry of protein-protein and protein-nucleic acid complexes. Stable isotopically labeled compounds are used to monitor the stable isotopomers in metabolism. Chapter 2 extends the knowledge of composition and dissociation pathways of the collision induced dissociation (CID) fragments of protonated Glu, Gln, Pro and Ala down to low m/z (m/z 27) range. The information provides a framework to monitor each carbon of the amino acids to explore the ammonia detoxification physiological metabolism. Native MS as a tool in structural biology can ionize native-like, noncovalent protein complexes. By combined native MS with tandem mass spectrometry (MS/MS) and ion mobility (IM), the quaternary structure and stoichiometry of protein complexes can be revealed. To develop a complementary activation method of CID, surface induced dissociation (SID) was applied to some refractory protein complexes that cannot be dissociated significantly in CID. SID successfully dissociates them to provide substructure information. Charge reduction and SID provide more compact fragments, which preserves more native structure information (Chapter 3). SID is also employed to dissociate 20S proteasome composed of four seven-member rings. The ring structures can be preserved in the SID fragments while CID produced monomers and the complementary (n-1)mer fragments (Chapter 4). Protein-nucleic acid complexes are more difficult to analyze using native MS because of heterogeneity of nucleic acids sequences and the requirement for nonvolatile salts to preserve the complexes. Chapter 5, 6 and 7 show that distinguishable MS peaks of protein-nucleic acid complexes can be achieved to provide stoichiometry information with careful optimization of the sample preparation, solution conditions, and instrument parameters. IM helps to extract peaks from the background noise. The active form of the endonuclease SgrAI is high molecular weight species (HMWS). Native MS and IM reveal that the basic building block is an SgrAI dimer with one copy of the double stranded DNA substrate. The conformation of the protein-DNA complex is discussed by comparing the experimental and theoretical collision cross section (Chapter 5). Another DNA/protein complex, the intact nucleosome, was ionized and showed significant covalent fragmentation in CID but not in SID (Chapter 6). In addition, the stoichiometry of the individual RNA and protein subunits in Pyrococcus furiosus RNase P was revealed for the first time by native MS and IM. Despite different oligomeric states reported for protein subunits not bound to the RNA, the ribonucleoprotein (RNP) complex has only one copy of each protein subunit. By examining the molecular mass of the RNP assembly intermediates and the final holoenzyme, we have also gained insight into its possible biogenesis route (Chapter 7). Successful applications of MS to study large biological macromolecular complexes illustrate that this is a powerful tool for bioanalytical chemistry, complementary to high-resolution methods such as NMR, X-ray crystallography and cryo-EM.
Vicki Wysocki, PhD (Advisor)
Venkat Gopalan, PhD (Committee Member)
Mark Foster, PhD (Committee Member)
310 p.
Biophysics; Chemistry
native mass spectrometry; ion mobility; surface induced dissociation; protein-protein; protein-nucleic acid; DNA; RNA

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Citations

  • Ma, X. (2014). MASS SPECTROMETRY DISSOCIATION STUDIES OF PROTEIN-PROTEIN AND PROTEIN-NUCLEIC ACID COMPLEXES AND 13C FLUX OF AMINO ACIDS [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397773864

    APA Style (7th edition)

  • Ma, Xin. MASS SPECTROMETRY DISSOCIATION STUDIES OF PROTEIN-PROTEIN AND PROTEIN-NUCLEIC ACID COMPLEXES AND 13C FLUX OF AMINO ACIDS. 2014. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1397773864.

    MLA Style (8th edition)

  • Ma, Xin. "MASS SPECTROMETRY DISSOCIATION STUDIES OF PROTEIN-PROTEIN AND PROTEIN-NUCLEIC ACID COMPLEXES AND 13C FLUX OF AMINO ACIDS." Doctoral dissertation, Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397773864

    Chicago Manual of Style (17th edition)

osu1397773864
262
© 2014, some rights reserved.Creative Commons License MASS SPECTROMETRY DISSOCIATION STUDIES OF PROTEIN-PROTEIN AND PROTEIN-NUCLEIC ACID COMPLEXES AND 13C FLUX OF AMINO ACIDS by Xin Ma is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by The Ohio State University and OhioLINK.