Characterization of phosphorylation sites on histone H1 isoforms by tandem mass spectrometry
Characterization of phosphorylation sites on histone H1 isoforms by tandem mass spectrometry
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DOI：
10.1021/pr0498887
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发表时间：
2004-11-01
影响因子：
4.4
通讯作者：
Hunt, DF
中科院分区：
文献类型：
--
作者：
Garcia, BA;Busby, SA;Hunt, DF
从异步生长的HeLa细胞中分离的组蛋白H1亚型进行酶消化，并通过纳米流反相高效液相色谱（RP-HPLC）串联质谱（MS/MS）在四极杆离子阱和线性四极杆离子阱傅立叶变换离子回旋共振质谱仪上进行分析。我们已经观察到组蛋白H1的所有五种主要亚型（H1.1，H1.2，H1.3，H1.4和H1.5）以及较少研究的H1亚型H1.X。MS/MS实验证实了所有亚型上的N-末端乙酰化加上单个内部乙酰化位点。采用固定化金属亲和色谱结合串联质谱法鉴定了5种主要H1亚型和H1.X上的19个磷酸化位点。这些磷酸化位点中的14个位于含有细胞周期蛋白依赖性激酶（CDK）共有基序（S/T）-P-X-Z（其中X是任何氨基酸，Z是碱性氨基酸）的肽上。五个磷酸化位点被确定在不符合共识CDK基序的区域。其中一个磷酸化位点位于H1.4肽KARKSAGAAKR的丝氨酸残基上。与磷酸丝氨酸相邻的赖氨酸残基也被甲基化。这一发现提出了一个问题，即假设的“甲基/磷”开关是否可以扩展到连接组蛋白，而不是唯一的核心组蛋白。
Histone H1 isoforms isolated from asynchronously grown HeLa cells were subjected to enzymatic digestion and analyzed by nano-flow reversed-phase high performance liquid chromatography (RP-HPLC) tandem mass spectrometry (MS/MS) on both quadrupole ion trap and linear quadrupole ion trap-Fourier transform ion cyclotron resonance mass spectrometers. We have observed all five major isoforms of histone H1 (H1.1, H1.2, H1.3, H1.4, and H1.5) as well as a lesser studied H1, isoform H1.X. MS/MS experiments confirmed N-terminal acetylation on all isoforms plus a single internal acetylation site. Immobilized metal affinity chromatography in combination with tandem mass spectrometry was utilized to identify 19 phosphorylation sites on the five major H1 isoforms plus H1.X. Fourteen of these phosphorylation sites were located on peptides containing the cyclin dependent kinase (CDK) consensus motif (S/T)-P-X-Z (where X is any amino acid and Z is a basic amino acid). Five phosphorylation sites were identified in regions that did not fit the consensus CDK motif. One of these phosphorylation sites was found on the serine residue on the H1.4 peptide KARKSAGAAKR. The adjacent lysine residue to the phosphoserine was also shown to be methylated. This finding raises the question of whether the hypothesized "methyl/phos" switch could be extended to linker histones, and not exclusive to core histones.