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Complete Trimethylation of Lysine Residues and its Application to the Quantitation of Lysine Methylation in Histones using Mass Spectrometry

Toth, Steven
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1430427165

Abstract Details

2015, Doctor of Philosophy, University of Toledo, College of Natural Sciences and Mathematics.
Post-translational modifications (PTMs) are an important step in protein biosynthesis. PTMs, like lysine methylation, occur frequently on histone proteins, the primary protein components of chromatin. The hydrophobic lysine methylation PTM can change the structure of the histone proteins which may affect its ability to recruit transcription factors. Abnormal levels of this modification have become good biomarkers for the proliferation of many diseases, like cancer. As these epigenetic changes are generally reversible, natural drug target methyltransferases and methyltransferase inhibitors have become popular treatments for these diseases. To study the efficacy of these drugs, a quantitative method for measuring lysine methylation levels is needed. There are many flaws to current methods for quantifying methylation: they are expensive, time-consuming, and can involve toxic reagents. Our method uses heavy isotopic labeled methyl groups to unilaterally convert all lysine residues to the trimethylated form, where they act as isotopomers to their natural counterparts that only differ by mass. We are able to quantify all levels of methylation and prove high precision and accuracy with the use of standards. Our method trimethylates protein lysine residues with the heavy-isotope methylating agents through a two-step process: complete dimethylation through the Eschweiler-Clarke reaction and complete trimethylation using methyl iodide. The natural, in vivo methylation will differ in mass from the heavy-isotope, in vitro chemical methyl addition by +3 Da for every heavy-isotope methyl group incorporated to the lysine residue. Mass spectrometric analyses were then performed to achieve absolute quantitation. Different levels of methylation were both observed and quantified in natural Bos taurus Histones H3 and H4. By universally converting all lysine residues to the trimethylated form without leaving any undermethylation or overmethylation (other residues methylated by these reagents), quantification of the different isotopomers could be achieved after correcting for any overlap from the previous isotopic series. Using this protocol, 100% coverage of mono- and dimethylated lysine residues was achieved on Bos taurus Histone H3 and Histone H4. There was 83% coverage of trimethylated residues on Histone H3. Various amounts of natural monomethylation were observed on lysine residues K9, K79, and K122 on Histone H3, with K79 also being naturally dimethylated and K9 also being di- and trimethylated. Quantitation was done at each residue as well. For example, lysine residue K9 was determined to be 27.58% ± 2.40% unmodified, 34.48% ± 4.41% monomethylated, 11.86% ± 3.24% dimethylated, and 27.58% ± 2.40% trimethylated. Quantification was confirmed to be accurate and precise by the use of standards that contained known levels of mono- and dimethylation on Histone H3 lysine residue K79 that were mixed it certain ratios. A mixture of 1:3 monomethylated gave a deviation of ±0.0200 from the expected value, and a standard deviation of ±0.0173. A mixture of 1:3 monomethylated gave a deviation of ±0.0567 from the expected value, and a standard deviation of ±0.0208. After analysis, the ratios were confirmed with a very low deviation, and repeated trials of different standard mixtures gave a similar variance proving that this quick and inexpensive quantification method is also reliable and repeatable.
Wendell Griffith, PhD (Committee Co-Chair)
Dragan Isailovic, PhD (Committee Co-Chair)
Jon Kirchhoff, PhD (Committee Member)
Amanda Bryant-Friedrich, PhD (Committee Member)
166 p.
Chemistry
Lysine methylation; Histones; Histone code; Lysine Methylation Quantitation

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Citations

  • Toth, S. (2015). Complete Trimethylation of Lysine Residues and its Application to the Quantitation of Lysine Methylation in Histones using Mass Spectrometry [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1430427165

    APA Style (7th edition)

  • Toth, Steven. Complete Trimethylation of Lysine Residues and its Application to the Quantitation of Lysine Methylation in Histones using Mass Spectrometry. 2015. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1430427165.

    MLA Style (8th edition)

  • Toth, Steven. "Complete Trimethylation of Lysine Residues and its Application to the Quantitation of Lysine Methylation in Histones using Mass Spectrometry." Doctoral dissertation, University of Toledo, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1430427165

    Chicago Manual of Style (17th edition)

toledo1430427165
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This open access ETD is published by University of Toledo and OhioLINK.