FCCC LOGO Faculty Publications
A quantitative multiplexed mass spectrometry assay for studying the kinetic of residue-specific histone acetylation
Methods. 2014 Dec;70(2-3) :127-33
PMID: 25123533    PMCID: PMC4477693    URL: http://www.ncbi.nlm.nih.gov/pubmed/25123533
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Abstract
Histone acetylation is involved in gene regulation and, most importantly, aberrant regulation of histone acetylation is correlated with major human diseases. Although many lysine acetyltransferases (KATs) have been characterized as being capable of acetylating multiple lysine residues on histones, how different factors such as enzyme complexes or external stimuli (e.g. KAT activators or inhibitors) alter KAT specificity remains elusive. In order to comprehensively understand how the homeostasis of histone acetylation is maintained, a method that can quantitate acetylation levels of individual lysines on histones is needed. Here we demonstrate that our mass spectrometry (MS)-based method accomplishes this goal. In addition, the high throughput, high sensitivity, and high dynamic range of this method allows for effectively and accurately studying steady-state kinetics. Based on the kinetic parameters from in vitro enzymatic assays, we can determine the specificity and selectivity of a KAT and use this information to understand what factors influence histone acetylation. These approaches can be used to study the enzymatic mechanisms of histone acetylation as well as be adapted to other histone modifications. Understanding the post-translational modification of individual residues within the histones will provide a better picture of chromatin regulation in the cell.
Notes
Kuo, Yin-Ming Henry, Ryan A Andrews, Andrew J ENG T32 CA009035/CA/NCI NIH HHS/ 2T32 CA-009037/CA/NCI NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't 2014/08/16 06:00 Methods. 2014 Dec;70(2-3):127-33. doi: 10.1016/j.ymeth.2014.08.003. Epub 2014 Aug 11.