Quantitative Mass Spectrometry Reveals that Intact Histone H1 Phosphorylations are Variant Specific and Exhibit Single Molecule Hierarchical Dependence
Quantitative Mass Spectrometry Reveals that Intact Histone H1 Phosphorylations are Variant Specific and Exhibit Single Molecule Hierarchical Dependence
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DOI：
10.1074/mcp.m114.046441
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发表时间：
2016-03-01
影响因子：
7
通讯作者：
Young, Nicolas L.
中科院分区：
文献类型：
--
作者：
Chen, Yu;Hoover, Michael E.;Young, Nicolas L.
2014年，乳腺癌是女性癌症相关死亡的第二大原因。最近的研究表明，组蛋白H1的磷酸化可能有助于作为乳腺癌和其他癌症的临床生物标记物，因为它能够识别增殖细胞群。虽然监测单个磷酸化的H1残基足以对高级别乳腺肿瘤进行分层，但扩大我们对H1如何在细胞周期中被磷酸化的知识对于了解其在癌症发生中的作用是至关重要的。由于大多数细胞表达的高度同源序列变体的存在，用自下而上的MS进行H1分析是具有挑战性的。这些高度碱性的蛋白质很难用LC-MS/MS进行分析，因为胰酶消化产生的多肽很小，具有亲水性。尽管自下而上的方法可以识别几个H1磷酸化事件，但这些多肽对于观察目标蛋白质上的组合翻译后修饰(PTM)模式并不有用。为了补充自下而上的MS提供的信息，我们利用自上而下的MS/MS工作流程来识别和定量与乳腺细胞在细胞周期中的进展相关的H1蛋白形式。在转移性乳腺细胞中观察到组蛋白H1.2和H1.4，而另一个组蛋白变体H1.3仅在非肿瘤性MCF-10A细胞中被发现。在有丝分裂(M期)的两个细胞系中都发现了组蛋白H1.4的进行性磷酸化。磷酸化首先发生在S172，然后依次是S187、T18、T146和T154。值得注意的是，H1.4的组蛋白H1.2和S172、S187、T18、T146和T154的S173位的磷酸化水平在M期显著高于S期，提示这些事件是细胞周期依赖性的，可能作为细胞增殖的标志。最后，我们报道了在MCF-10A细胞中观察到的H1.2SNP变异体A18V。
Breast cancer was the second leading cause of cancer related mortality for females in 2014. Recent studies suggest histone H1 phosphorylation may be useful as a clinical biomarker of breast and other cancers because of its ability to recognize proliferative cell populations. Although monitoring a single phosphorylated H1 residue is adequate to stratify high-grade breast tumors, expanding our knowledge of how H1 is phosphorylated through the cell cycle is paramount to understanding its role in carcinogenesis. H1 analysis by bottom-up MS is challenging because of the presence of highly homologous sequence variants expressed by most cells. These highly basic proteins are difficult to analyze by LC-MS/MS because of the small, hydrophilic nature of peptides produced by tryptic digestion. Although bottom-up methods permit identification of several H1 phosphorylation events, these peptides are not useful for observing the combinatorial post-translational modification (PTM) patterns on the protein of interest. To complement the information provided by bottom-up MS, we utilized a top-down MS/MS workflow to permit identification and quantitation of H1 proteoforms related to the progression of breast cells through the cell cycle. Histones H1.2 and H1.4 were observed in MDA-MB-231 metastatic breast cells, whereas an additional histone variant, histone H1.3, was identified only in nonneoplastic MCF-10A cells. Progressive phosphorylation of histone H1.4 was identified in both cell lines at mitosis (M phase). Phosphorylation occurred first at S172 followed successively by S187, T18, T146, and T154. Notably, phosphorylation at S173 of histone H1.2 and S172, S187, T18, T146, and T154 of H1.4 significantly increases during M phase relative to S phase, suggesting that these events are cell cycle-dependent and may serve as markers for proliferation. Finally, we report the observation of the H1.2 SNP variant A18V in MCF-10A cells.