Abstract
Histone proteins are subject to a range of post-transcriptional modifications in living cells. The combinatorial nature of these modifications constitutes the "histone code" that dictates chromatin structure and function during development, growth, differentiation, and homeostasis of cells. Deciphering of the histone code is hampered by the lack of analytical methods for monitoring the combinatorial complexity of reversible multisite modifications of histones, including acetylation and methylation. To address this problem, we used LC-MSMS technology and Virtual Expert Mass Spectrometrist software for qualitative and quantitative proteomic analysis of histones extracted from human small cell lung cancer cells. A total of 32 acetylations, methylations, and ubiquitinations were located in the human histones H2A, H2B, H3, and H4, including seven novel modifications. An LC-MSMS-based method was applied in a quantitative proteomic study of the dose-response effect of the histone deacetylase inhibitor (HDACi) PXD101 on histone acetylation in human cell cultures. Triplicate LC-MSMS runs at six different HDACi concentrations demonstrated that PXD101 affects acetylation of histones H2A, H2B, H3, and H4 in a site-specific and dose-dependent manner. This unbiased analysis revealed that a relative increase in acetylated peptide from the histone variants H2A, H2B, and H4 was accompanied by a relative decrease of dimethylated Lys(57) from histone H2B. The dose-response results obtained by quantitative proteomics of histones from HDACi-treated cells were consistent with Western blot analysis of histone acetylation, cytotoxicity, and dose-dependent expression profiles of p21 and cyclin A2. This demonstrates that mass spectrometry-based quantitative proteomic analysis of post-translational modifications is a viable approach for functional analysis of candidate drugs, such as HDAC inhibitors.
Highlights
Histone proteins are subject to a range of post-transcriptional modifications in living cells
There is a great interest in developing histone deacetylase inhibitor (HDACi) as anticancer drugs, and some of these compounds are currently being evaluated in clinical trails [16, 17]
If the q-value is above a critical value, determined to be 4.75 (q␣, 12, 6 where ␣ ϭ 0.05 is the significance level, 12 is the number of degrees of freedom, and 6 is the number of groups) the sampled group is significantly different from the group corresponding to 0 M HDACi
Summary
Cell Culture—Human small cell lung cancer carcinoma OC-NYH cells have been described previously [23]. Quantification of peptides carrying modified arginine and/or lysine residues was performed by using the features of the VEMS Version 3.0 software [26, 27] These features included protein identification, PTM assignment, and alignment and calibration of LC-MSMS data with respect to peptide retention times and peptide masses. If the q-value is above a critical value, determined to be 4.75 (q␣, 12, 6 where ␣ ϭ 0.05 is the significance level, 12 is the number of degrees of freedom, and 6 is the number of groups) the sampled group is significantly different from the group corresponding to 0 M HDACi. Significance analysis of microarrays (SAM) analysis was performed to identify peptides that showed significant abundance changes over the different concentrations of HDACi. The SAM analysis tests the overall significance of difference in peptide peak intensity in the different runs by compensating for multiple testing [31]
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