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Characterization of Histone H1 and Extracellular Vesicles by Mass Spectrometry

Harshman, Sean William
http://rave.ohiolink.edu/etdc/view?acc_num=osu1385544818

Abstract Details

2013, Doctor of Philosophy, Ohio State University, Integrated Biomedical Science Graduate Program.
The use of mass spectrometry to analyze difficult samples and to identify large numbers of proteins has been used in many applications. Utilizing mass spectrometric approaches, I first developed a method for isolation and analysis of histone H1 from different cellular compartments. This study showed that linker histones enriched by cellular fractionation gives less nuclear contamination and higher histone content than when prepared by nuclei isolation. Utilizing the developed method, the soluble linker histones were profiled throughout the cell cycle revealing phosphorylation increases as cells reach mitosis. To demonstrate the utility of the analysis, histone H1.2-H1.5 translocation to the cytosol was monitored in response to the CDK inhibitor flavopiridol in primary CLL cells treated ex vivo. Data shows all H1 variants translocate in response to drug treatment with no specific order to their cytosolic appearance. The results illustrate the utility of cellular fractionation in conjunction with LC-MS for the analysis of histone H1 throughout the cell. Next, I characterized the histone H1 phosphorylation from breast cancer cells. The results from this study show the extent of H1 phosphorylation can distinguish between the different cell lines. The phosphorylation at threonine 146 was found to be a site of mitotic histone H1 phosphorylation with potential biological implications. To explore the prognostic value of H1 phosphorylation, extracellular stimulus, estradiol or kinase inhibitor LY294002, were applied to cells in vitro to monitor changes in histone H1 phosphorylation. The data show that histone H1 phosphorylation (T146) can increase and decrease in response to the extracellular stimuli applied. Further exploration of this hypothesis was conducted by staining primary breast tumors for T146 phosphorylation. Variable staining patterns were observed across tumor grades and subtypes with pT146 labeling and associate with tumor grade. These results establish the potential for histone H1 phosphorylation at threonine 146 as a clinical biomarker in breast cancer. Finally, I identify the protein content of extracellular vesicles isolated from multiple myeloma (MM) cell lines. These two studies show the protein content and relative abundance of vesicles derived from four distinct MM cell lines. Additionally the localization of serum CD44 is shown to be on the peripheral vesicles of MM patients. The utility of serum CD44 as a biomarker in MM is shown through positive correlation with the accepted MM biomarker beta 2-microglobulin. These results generate a foundation for hypothesis driven studies of MM biology and the potential use of serum CD44 as a novel serum biomarker of MM. Collectively, our results utilize mass spectrometric approaches to solve biological problems across two distinct disease states.
Michael Freitas, Ph.D (Advisor)
Jeffrey Parvin, M.D., Ph.D (Committee Member)
Mark Parthun, Ph.D (Committee Member)
Mitch Phelps, Ph.D (Committee Member)
424 p.
Biomedical Research
Mass Spectrometry; Histone H1; Biomarker; Exosomes; Microvesicles; Extracellular Vesicles; Shotgun Proteomics

Recommended Citations

Citations

  • Harshman, S. W. (2013). Characterization of Histone H1 and Extracellular Vesicles by Mass Spectrometry [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1385544818

    APA Style (7th edition)

  • Harshman, Sean. Characterization of Histone H1 and Extracellular Vesicles by Mass Spectrometry. 2013. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1385544818.

    MLA Style (8th edition)

  • Harshman, Sean. "Characterization of Histone H1 and Extracellular Vesicles by Mass Spectrometry." Doctoral dissertation, Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1385544818

    Chicago Manual of Style (17th edition)

osu1385544818
153
© 2013, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.