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Protein Primary and Quaternary Structure Elucidation by Mass Spectrometry

Song, Yang
http://rave.ohiolink.edu/etdc/view?acc_num=osu1437649750

Abstract Details

2015, Doctor of Philosophy, Ohio State University, Chemistry.
Mass spectrometry (MS) has become an indispensable tool for characterizing proteins, from primary structures to quaternary structures. This dissertation shows efforts to expand the role of MS in protein characterization. Chapter 2 is focused on protein primary structure determination. Blood collected from 33 bird species that are common in the U.S. as hosts for ticks but that have unreported hemoglobin sequences was provided to the Wysocki group. The sequence information for the bird hemoglobin is important for tick-borne disease prevention and intervention. A top-down-assisted bottom-up MS approach with a customized searching database, based on variability in known bird hemoglobin sequences, was devised to facilitate fast and complete sequencing of hemoglobin from birds with unknown sequences and hemoglobin from all 33 species was sequenced. Besides sequencing primary structures of protein complexes in denaturing conditions, MS can be utilized to study intact non-covalent protein complexes in the gas phase, retaining memory of their solution phase structures. Tandem MS is applied to dissociate non-covalent protein complexes, probing their subunit connectivity by generating structurally informative subcomplexes. The most widely used gas-phase dissociation method is collision induced dissociation (CID), which generally does not provide enough substructural information. An alternative gas-phase disassembly method, surface induced dissociation (SID), has been shown to reveal substructure information. Especially in recent years, with the incorporation of an SID device into a Waters SYNAPT G2 or G2-S mass spectrometer with ion mobility capability, the conformation of SID products and remaining precursor were studied to improve our understanding of the SID process. In Chapter 3, 4 and 5, fundamental studies on the gas-phase dissociation behavior of homodimers (enolase, a-lactalbumin, and ß-lactoglobulin), inter-chain disulfide reduced monoclonal antibody, and homo-hexamer protein complexes (bovine GDH, bacterial GDH, GCH and insulin) are performed. SID has been shown to be beneficial for protein complex interface analysis, because SID minimizes intra-protein conformational disruptions (unfolding) of subunits in the dissociation process. In addition, an excellent correlation of the SID behaviors and in silico analysis of interfacial areas of known systems demonstrates that SID can be very useful in structural characterization of unknown systems, especially in predicting the interfaces and relative interfacial strength. Multiple MS based approaches were then utilized to predict quaternary structure of toyocamycin nitrile hydratase (TNH) (Chapter 6). Unfortunately, there are no structures solved by either NMR or x-ray crystallography on this important enzymatic complex. By coupling SID with ion mobility mass spectrometry (SID/IM), a complete connectivity map with relative interfacial strengths is obtained. Collisional cross-sections (CCS) measured from IM experiments are used as constraints for postulating a coarse-grained complex model. Protein complex homology modeling with all the constraints from connectivity, CCS and covalent labeling is utilized to propose likely structures with atomic coordinates for TNH hexamer. A similar comprehensive mass spectrometric structural analysis was performed in Chapter 8 on a multicopper oxidase protein complex that is has no crystal structure or NMR results due to its heterogeneity and large size (over 200 kDa). Overall, this dissertation has expanded the role of MS in protein characterization.
Vicki Wysocki, Dr. (Advisor)
Mark Foster, Dr. (Committee Member)
Abraham Badu-Tawiah, Dr. (Committee Member)
265 p.
Analytical Chemistry; Biophysics
Mass spectrometry; Protein sequencing; Bottom-up; Top-down; Customized database; Hemoglobin; Surface induced dissociation; Toyocamycin Nitrile Hydratase; Ion mobility; Coarse-grained modeling; Surface mapping

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Citations

  • Song, Y. (2015). Protein Primary and Quaternary Structure Elucidation by Mass Spectrometry [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1437649750

    APA Style (7th edition)

  • Song, Yang. Protein Primary and Quaternary Structure Elucidation by Mass Spectrometry. 2015. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1437649750.

    MLA Style (8th edition)

  • Song, Yang. "Protein Primary and Quaternary Structure Elucidation by Mass Spectrometry." Doctoral dissertation, Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1437649750

    Chicago Manual of Style (17th edition)

osu1437649750
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This open access ETD is published by The Ohio State University and OhioLINK.