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Development of a Mass Spectrometry-Based Method for the Quantitation of Lysine Methylation

Berardinelli, Anthony Michael
http://orcid.org/0000-0002-8768-2520
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1483710924985294

Abstract Details

2017, Master of Science, University of Toledo, Chemistry.
Protein methylation affects processes such as X-chromosome inactivation, gene expression and telomere position effects as well as the activity of chromatin domains. The ability to measure methylation quantitatively would allow for the characterization of differences in methylation states of proteins in various diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, breast cancer and Parkinson’s disease. The main goal of this thesis work was to develop mass spectrometry-based analytical methods for quantitation of lysine methylation in proteins. A two-step methylation procedure was developed using L-lysine, further optimized with peptides leucine-serine-lysine-leucine-NH2 (LSKL-NH2) and arginine-lysine-glutamic acid-valine-tyrosine acetate salt (RKEVY-OH), and ubiquitin. Optimization of the two-step procedure involved examining the quantities of methylating reagents required, buffer used, reagent and buffer removal, pH of the reaction and in-gel versus in-solution methylation. The modifications were accomplished by the synthetic trimethylation by chemical synthesis of the e-amine side chain of lysine and the characterization of the chemical synthesis was done using stable-isotope labeled compounds. Stable isotope-labeled compounds and both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry instrumentation were used for the quantitative proteomics of the histone proteins, while ESI-MS was used for the peptide studies.
Max Funk (Committee Co-Chair)
Wendell Griffith (Committee Co-Chair)
Dragan Isailovic (Committee Member)
Amanda Bryant-Friedrich (Committee Member)
168 p.
Analytical Chemistry; Biochemistry; Chemistry
Trimethylation, amine, chemical trimethylation, quantitative chemical methylation, protein lysine methylation, deuterated analogs , heavy labeling

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Citations

  • Berardinelli, A. M. (2017). Development of a Mass Spectrometry-Based Method for the Quantitation of Lysine Methylation [Master's thesis, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1483710924985294

    APA Style (7th edition)

  • Berardinelli, Anthony. Development of a Mass Spectrometry-Based Method for the Quantitation of Lysine Methylation. 2017. University of Toledo, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1483710924985294.

    MLA Style (8th edition)

  • Berardinelli, Anthony. "Development of a Mass Spectrometry-Based Method for the Quantitation of Lysine Methylation." Master's thesis, University of Toledo, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1483710924985294

    Chicago Manual of Style (17th edition)

toledo1483710924985294
337
© 2011, some rights reserved.Creative Commons License Development of a Mass Spectrometry-Based Method for the Quantitation of Lysine Methylation by Anthony Michael Berardinelli 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 University of Toledo and OhioLINK.