The methylation of histone lysine residues has been correlated to numerous phenotypes of cancer. Histone lysine residues can have up to three methyls added and each state can have clearly different cellular roles. For years, researchers in epigenetics (including those focused on epigenetic processes in cancer) have used techniques that rely on antibodies or radioactivity to measure the methylation state of a given lysine;however, these are neither comprehensive nor quantitative. What has become an alternative approach to get around these limitations is the direct use of mass spectrometry, but the problem with this approach is that differentially methylated peptides do not ionize the same and CANNOT be directly compared as a measurement of activity. Therefore, the field of cancer epigenetic research and epigenetics as a whole needed a comprehensive method to simultaneously monitor demethylase/methyltransferase reaction intermediates (i.e., different methyl states on a lysine) in a quantitative manner. In 2011, we provided a novel approach for the comprehensive and quantitative measurement of lysine methylation states, which is called MassSQUIRM (Mass Spectrometric Quantitation Using Isotopic Reductive Methylation). MassSQUIRM utilizes the chemical incorporation of isotopically heavy methyl groups on lysines to convert all reaction intermediates (un- and monomethyl) to fully dimethyl lysines (differing only by hydrogen and deuterium - which does not affect ionization properties in mass spectrometry). A comparison of peptide intensities of the mixture of heavy and light species allows for comprehensive (un-, mono- and dimethyl states) quantitation of lysine methylation. We recently published the MassSQUIRM technique, and in this application we outline how we will evaluate it in a cancer relevant context in order to ultimately develop a kit for cancer research. Our overall goal is to provide a MassSQUIRM kit to cancer researchers to assay demethylation and methylation (un-, mono- and dimethylation specifically) of lysine residues in proteins correlated to particular cancer phenotypes. To validate the MassSQUIRM approach for its use in cancer research, we will pursue the following Aims:
Aim 1. Determine the general applicability of MassSQUIRM by assaying a panel of histone lysine demethylases and methyltransferases.
Aim 2. Evaluate the effectiveness of using MassSQUIRM to assay LSD1 activity from cell lysates.
Aim 3. Optimize MassSQUIRM for lysine demethylation screening with a panel of LSD1 inhibitors.

Public Health Relevance

The posttranslational modification of histones has been correlated to numerous cancer phenotypes. The field of cancer research focusing on histone epigenetic regulation has been hampered by techniques to quantitatively and comprehensively study one of these histone posttranslational modifications, namely lysine methylation. Recently we published a technique, MassSQUIRM, that overcomes these limitations. In the proposed work, we will expand on the utility of MassSQUIRM to make it broadly useful for cancer researchers.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Exploratory/Developmental Grants Phase II (R33)
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Special Emphasis Panel (ZCA1)
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Knowlton, John R
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University of Arkansas for Medical Sciences
Schools of Medicine
Little Rock
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Heard, Melissa E; Besio, Roberta; Weis, MaryAnn et al. (2016) Sc65-Null Mice Provide Evidence for a Novel Endoplasmic Reticulum Complex Regulating Collagen Lysyl Hydroxylation. PLoS Genet 12:e1006002
Salinas, Eduardo; Byrum, Stephanie D; Moreland, Linley E et al. (2016) Identification of Viral and Host Proteins That Interact with Murine Gammaherpesvirus 68 Latency-Associated Nuclear Antigen during Lytic Replication: a Role for Hsc70 in Viral Replication. J Virol 90:1397-413
Sengupta, Deepanwita; Byrum, Stephanie D; Avaritt, Nathan L et al. (2016) Quantitative Histone Mass Spectrometry Identifies Elevated Histone H3 Lysine 27 (Lys27) Trimethylation in Melanoma. Mol Cell Proteomics 15:765-75
Sengupta, Deepanwita; Tackett, Alan J (2016) Proteomic Findings in Melanoma. J Proteomics Bioinform 9:
Blair, Lauren P; Liu, Zongzhi; Labitigan, Ramon Lorenzo D et al. (2016) KDM5 lysine demethylases are involved in maintenance of 3'UTR length. Sci Adv 2:e1501662
Atanassov, Boyko S; Mohan, Ryan D; Lan, Xianjiang et al. (2016) ATXN7L3 and ENY2 Coordinate Activity of Multiple H2B Deubiquitinases Important for Cellular Proliferation and Tumor Growth. Mol Cell 62:558-71
Byrum, Stephanie D; Burdine, Marie S; Orr, Lisa et al. (2016) A Quantitative Proteomic Analysis of Urine from Gamma-Irradiated Non-Human Primates. J Proteomics Bioinform 9:
Ayyadevara, Srinivas; Mercanti, Federico; Wang, Xianwei et al. (2016) Age- and Hypertension-Associated Protein Aggregates in Mouse Heart Have Similar Proteomic Profiles. Hypertension 67:1006-13
Haun, Randy S; Quick, Charles M; Siegel, Eric R et al. (2015) Bioorthogonal labeling cell-surface proteins expressed in pancreatic cancer cells to identify potential diagnostic/therapeutic biomarkers. Cancer Biol Ther 16:1557-65
Byrum, Stephanie D; Taverna, Sean D; Tackett, Alan J (2015) Purification of specific chromatin loci for proteomic analysis. Methods Mol Biol 1228:83-92

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