Abstract
Global histone modifications and their putative relevance to short and long term cellular programming have drawn substantial interest in the study of chromatin. Here we describe the use of reverse-phase liquid chromatography coupled to Linear Ion Trap-Fourier Transform Mass Spectrometry (RPLC-LTQ-FTMS) to quickly profile post-translationally modified isoforms and variants for core histone proteins from as few as 5 × 104 cells at isotopic resolution. Such LC-MS profiling greatly facilitated the detection of histones from HeLa S3 or 293T cells experiencing shRNA- or siRNA-knockdown of histone deacetylase (HDAC) 1, 2, 3 or 1 and 2 together. In no case was significant global histone hyperacetylation relative to control cells observed, suggesting possible compensation of deacetylation activity by partially redundant enzymes in the 18-member HDAC family. This contrasts sharply with yeast where genetic deletion of HDAC rpd3 causes massive hyperacetylation. Treatment of cells with TSA and class I selective HDAC inhibitors had similar ability to induce global histone hyperactylation, though to different extents in HeLa S3 vs. 293T cells.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 3885-3892 |
| Number of pages | 8 |
| Journal | Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences |
| Volume | 877 |
| Issue number | 30 |
| DOIs | |
| State | Published - Nov 15 2009 |
Funding
The authors thank Dr. Jie Chen for pCMV-dR8.91 and pCMV-VSV-G plasmids for shRNA knockdown experiments. This work was supported by generous grants from the Packard Foundation and the National Institutes of Health ( GM 067193-06 ) to N.L.K.
Keywords
- Acetylation
- HDACs
- Histone
- LC-FTMS
ASJC Scopus subject areas
- Analytical Chemistry
- Biochemistry
- Clinical Biochemistry
- Cell Biology