Department of Biology, Saint Mary's College of California, Moraga, CA 94575, United States.
QB3/Chemistry Mass Spectrometry Facility, University of California, Berkeley, CA 94720, United States.
Methods. 2020 Dec 1;184:102-111. doi: 10.1016/j.ymeth.2020.01.019. Epub 2020 Jan 31.
The development of universal, broadly applicable methods for histone extraction from animal cells and tissues has unlocked the ability to compare these epigenetic-influencing proteins across tissue types, healthy and diseased states, and cancerous versus normal cells. However, for plants and green algae, a quick and easily implemented histone extraction method has yet to be developed. Here, we report an optimized method that provides a unified approach to extract histones for the green microalgal species Chlamydomonas reinhardtii and Scenedesmus dimorphus as well as for maize (corn) leaf tissue. Histone extraction methods include treatment with high salt concentrations and acidification. Preparations of nuclei can be made in ∼3.5 h and histones extracted in ∼3.5 h either immediately or nuclei may be frozen and histone proteins can be later extracted without a change in histone PTM patterns. To examine the efficiency of the new methods provided, we performed both qualitative and quantitative analysis of salt and acid-extracted whole histone proteins (SAEWH) via SDS-PAGE gel electrophoresis and intact protein mass spectrometry. SDS-PAGE analysis indicated that histone yields decrease when using walled Chlamydomonas strains relative to cell-wall-less mutants. Using top-down mass spectrometry (TDMS) for intact protein analysis, we confirmed the presence of H4K79me1 in multiple algal species; however, this unique modification was not identified in corn leaf tissue and has not been reported elsewhere. TDMS measurements of SAEWH extracts also revealed that oxidation which occurs during the histone extraction process does not increase with exposure of harvested algal cells, their nuclei and the extracted histone samples to light.
从动物细胞和组织中提取组蛋白的通用、广泛适用的方法的发展，使得比较不同组织类型、健康和疾病状态以及正常细胞与癌细胞之间的这些表观遗传影响蛋白成为可能。然而，对于植物和绿藻来说，一种快速且易于实施的组蛋白提取方法尚未被开发出来。在这里，我们报告了一种优化的方法，该方法为绿藻小球藻和杜氏盐藻以及玉米（玉米）叶片组织提供了一种统一的提取组蛋白的方法。组蛋白提取方法包括高盐浓度和酸化处理。核的制备可以在大约 3.5 小时内完成，组蛋白可以在大约 3.5 小时内立即提取，或者可以将核冷冻，以后再提取组蛋白，而不会改变组蛋白 PTM 模式。为了检验新方法的效率，我们通过 SDS-PAGE 凝胶电泳和完整蛋白质质谱法对盐和酸提取的全组蛋白（SAEWH）进行了定性和定量分析。SDS-PAGE 分析表明，与无细胞壁突变体相比，使用有细胞壁的衣藻菌株时，组蛋白产量会降低。使用自上而下的质谱法（TDMS）对完整蛋白质进行分析，我们在多种藻类物种中证实了 H4K79me1 的存在；然而，这种独特的修饰在玉米叶片组织中没有被发现，也没有在其他地方报道过。SAEWH 提取物的 TDMS 测量还表明，在组蛋白提取过程中发生的氧化不会随着收获的藻类细胞、它们的核和提取的组蛋白样品暴露在光线下而增加。
