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Biochemical and MALDI-MS Methods for Characterization of Ribosomal Proteins

Hamburg, Daisy-Malloy
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1204305343

Abstract Details

2008, PhD, University of Cincinnati, Arts and Sciences : Chemistry.
The goals of this dissertation are to further the field of analytical chemistry by developing, optimizing and implementing new tools for ribosomal protein analysis and to further the field of ribosome structure/function determination. The main analytical tool used in this work is matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and the focus of the studies are ribosomal proteins. To understand how ribosomal proteins and ribosomal ribonucleic acids interact during protein synthesis, detailed knowledge of the structure of the ribosome is compulsory. My research focuses on the study of ribosomal proteins. The major theme throughout this dissertation is the development of MALDI-MS as a technique to identify proteins. The first study probes the reliability of the MALDI technique for identifying proteins whose sequences are not known. The MALDI-MS method is useful in distinguishing small changes among one or more proteins in a mixture and was used in several collaborative efforts, including characterization of Thermus thermophilus ribosomal proteins S12 and THX and Rattus norvegicus ribosomal proteins. Another major effort in this dissertation was to optimize a method combining limited proteolysis with MALDI-MS for the analysis of bacterial ribosomal proteins. The advantage of the limited proteolysis method is that it provides insight into the structure and enzymatic activity of ribosomal proteins with ribosomes and ribosomal subunits, and MALDI-MS analysis can be performed quickly on many samples. I discovered that the main sources of variability in these analyses arise from the enzymatic digestion and MALDI analysis steps in this method. I also investigated the hydrolysis-accessible regions of bacterial ribosomal proteins from Escherichia coli. I established that protease accessibility of ribosomal proteins in isolated subunits differs from that of intact ribosomes. The work presented in this dissertation is suitable for application to more complex organisms whose crystal structure information is not available. Specifically, MALDI-MS and limited proteolysis can be applied to analysis of yeast and/or other eukaryotic organisms to obtain information on the structures of these ribosomes. This work has furthered the field of ribosome analysis by providing methods that are suitable for obtaining information on ribosomal proteins involved in the complex structure of the ribosome.
Patrick Limbach, PhD (Committee Chair)
Thomas Beck, PhD (Committee Member)
Joseph Caruso, PhD (Committee Member)
190 p.
Analytical Chemistry; Biochemistry
MALDI-MS; ribosome; ribosomal proteins

Recommended Citations

Citations

  • Hamburg, D.-M. (2008). Biochemical and MALDI-MS Methods for Characterization of Ribosomal Proteins [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1204305343

    APA Style (7th edition)

  • Hamburg, Daisy-Malloy. Biochemical and MALDI-MS Methods for Characterization of Ribosomal Proteins. 2008. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1204305343.

    MLA Style (8th edition)

  • Hamburg, Daisy-Malloy. "Biochemical and MALDI-MS Methods for Characterization of Ribosomal Proteins." Doctoral dissertation, University of Cincinnati, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1204305343

    Chicago Manual of Style (17th edition)

ucin1204305343
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© 2008, all rights reserved.
This open access ETD is published by University of Cincinnati and OhioLINK.