Comprehensive phosphoprotein analysis of linker histone H1 from Tetrahymena thermophila
Comprehensive phosphoprotein analysis of linker histone H1 from Tetrahymena thermophila
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DOI：
10.1074/mcp.m600086-mcp200
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发表时间：
2006-09-01
影响因子：
7
通讯作者：
Hunt, Donald F.
中科院分区：
文献类型：
--
作者：
Garcia, Benjamin A.;Joshi, Swati;Hunt, Donald F.
连接体组蛋白H1在正常生长的嗜热四膜虫中高度磷酸化，但在某些条件下（如长时间饥饿）变得明显去磷酸化。由于H1的磷酸化与基因表达、DNA修复和其他关键过程的调控有关，我们试图使用基于质谱的方法来获得这种连接体组蛋白的深度磷酸化“特征”。组蛋白H1从生长和饥饿四膜虫进行了分析，通过纳流反相HPLC MS/MS酶消化，丙酸酐衍生化，磷酸肽富集通过IMAC。我们确认了五个磷酸化位点，并检测到两个新的磷酸化位点和两个新的乙酰化位点。H1上的磷酸化的顺序推导出通过使用质谱法来定义通过阳离子交换色谱分离的磷酸化H1亚型上的修饰位点。使用稳定同位素标记，IMAC和串联质谱法的组合，获得了从生长和饥饿的四膜虫分离的H1上的位点特异性磷酸化的相对水平。
Linker histone H1 is highly phosphorylated in normal growing Tetrahymena thermophila but becomes noticeably dephosphorylated in response to certain conditions such as prolonged starvation. Because phosphorylation of H1 has been associated with the regulation of gene expression, DNA repair, and other critical processes, we sought to use mass spectrometry-based approaches to obtain an in depth phosphorylation "signature" for this linker histone. Histone H1 from both growing and starved Tetrahymena was analyzed by nanoflow reversed-phase HPLC MS/MS following enzymatic digestions, propionic anhydride derivatization, and phosphopeptide enrichment via IMAC. We confirmed five phosphorylation sites identified previously and detected two novel sites of phosphorylation and two novel minor sites of acetylation. The sequential order of phosphorylation on H1 was deduced by using mass spectrometry to define the modified sites on phosphorylated H1 isoforms separated by cation-exchange chromatography. Relative levels of site-specific phosphorylation on H1 isolated from growing and starved Tetrahymena were obtained using a combination of stable isotopic labeling, IMAC, and tandem mass spectrometry.