Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH, USA; Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.
J Proteomics. 2013 Oct 8;91:595-604. doi: 10.1016/j.jprot.2013.08.022. Epub 2013 Sep 5.
Analysis of histones, especially histone H1, is severely limited by immunological reagent availability. This paper describes the application of cellular fractionation with LC-MS for profiling histones in the cytosol and upon chromatin. First, we show that linker histones enriched by cellular fractionation gives less nuclear contamination and higher histone content than when prepared by nuclei isolation. Second, we profiled the soluble linker histones throughout the cell cycle revealing phosphorylation increases as cells reach mitosis. Finally, we monitored histone H1.2-H1.5 translocation to the cytosol in response to the CDK inhibitor flavopiridol in primary CLL cells treated ex vivo. Data shows that all H1 variants translocate in response to drug treatment with no specific order to their cytosolic appearance. The results illustrate the utility of cellular fractionation in conjunction with LC-MS for the analysis of histone H1 throughout the cell.
This paper demonstrates the first time application of cellular fractionation to characterize cytosolic histone H1 by liquid chromatography mass spectrometry (LC-MS). Using the Ramos Burkitt's lymphoma cell line, cellular fractionation was shown to give less nuclear contamination and higher histone content than preparations by nuclei isolation. Further application of the cellular fractionation approach was shown by using primary chronic lymphocytic leukemia (CLL) cells to monitor the movement of histone H1 across cellular compartments in response to the cyclin dependent kinase inhibitor flavopiridol. Collectively, these data establish a mass spectrometric method for exploration into the function of cytosolic histone H1.
分析组蛋白，特别是组蛋白 H1，受到免疫试剂可用性的严重限制。本文描述了使用细胞分级分离与 LC-MS 对细胞质和染色质中组蛋白进行分析的应用。首先，我们表明，通过细胞分级分离富集连接组蛋白比通过核分离制备时具有更少的核污染和更高的组蛋白含量。其次，我们在整个细胞周期中对可溶性连接组蛋白进行了分析，结果表明，随着细胞进入有丝分裂，磷酸化增加。最后，我们监测了 CDK 抑制剂 flavopiridol 处理的原代 CLL 细胞中组蛋白 H1.2-H1.5 向细胞质的易位。数据表明，所有 H1 变体都在药物治疗下发生易位，而没有特定的顺序出现在细胞质中。结果表明，细胞分级分离与 LC-MS 结合用于分析整个细胞中的组蛋白 H1 的实用性。
本文首次应用细胞分级分离与液相色谱质谱联用（LC-MS）对细胞质组蛋白 H1 进行了特征分析。使用 Ramos 伯基特淋巴瘤细胞系，细胞分级分离比通过核分离制备具有更少的核污染和更高的组蛋白含量。进一步应用细胞分级分离方法，使用原代慢性淋巴细胞白血病（CLL）细胞监测组蛋白 H1 在细胞区室之间移动对细胞周期依赖性激酶抑制剂 flavopiridol 的反应。总的来说，这些数据建立了一种用于探索细胞质组蛋白 H1 功能的质谱方法。
