利用亲和层析法检测乙酰化蛋白的研究
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摘要
蛋白乙酰化与肿瘤的发生发展有密切关系,由乙酰化转移酶引起的蛋白乙酰化逐渐被认为是一种重要的生物调控修饰。乙酰化状态取决于组蛋白乙酰基转移酶(histone acetyltransferase,HATs)与组蛋白去乙酰化酶(histone deacetylase,HDACs)之间的活性竞争。
HDAC抑制剂会使染色质组蛋白乙酰化水平提高,因此导致特定基因激活表达,相应地导致细胞的末端分化或癌细胞的凋亡。初步的临床研究表明,人类可以安全地通过抑制HDAC的活性来获得组蛋白高乙酰化水平,HDACs已经成为抗癌新药开发的热门靶点,而研发这一类抗癌新药的前条件之一就是要建立适应于药物筛选所用快速、相对定量检测乙酰化蛋白方法。
目的：旨在建立一种快速、相对定量检测乙酰化蛋白方法,并初步探讨其应用。
方法：本文用乙酰化蛋白标准品寻找最优的质谱前处理方案,并将其应用于检测乙酰化蛋白方法的建模。模型建立如下：不同浓度的曲古菌素A(Trichostatin A.TSA)于不同的时间段刺激不同数量的Jurkat细胞,诱导其蛋白高乙酰化后,使用抗乙酰化赖氨酸抗体亲和层析柱富集纯化乙酰化总蛋白,经酸洗脱后固定于酶标板,ELISA检测乙酰化蛋白相对总量,并用MALDI-TOF-TOF分析验证其成分。然后利用该方法初步检测不同浓度的没食子酸、大黄素、单乙酰化大黄素A作用Jurkat细胞后乙酰化总蛋白水平的变化。
结果：1μmol/L的TSA作用于4×105Jurkat细胞24h,乙酰化总蛋白相对水平最高。MALDI-TOF-TOF分析显示,TSA诱导Jurkat细胞产生的乙酰化蛋白共有22种,其中15种为乙酰化组蛋白。
没食子酸、大黄素、单乙酰化大黄素A作用于Jurkat细胞后所导致的乙酰化程度不同,以1μmol/L浓度TSA为阳性对照组,无药物为空白对照组,35.09μmol/L和17.54μmol/L大黄素处理的Jurkat细胞蛋白乙酰化相对水平分别为4.3%和14.2%；1.47μmol/L和2.94μmol/L没食子酸处理组相对蛋白乙酰化水平分别为28.7%和11.5%；152.91μmol/L和30.58μmol/L单乙酰化大黄素组分别为22.0%和3.6%。其中1.47μmol/L没食子酸所诱导的乙酰化水平最高。
结论：初步建立了活细胞基础上纯化富集并检测乙酰化总蛋白水平的方法,该法可快速、简便的筛选以组蛋白去乙酰化酶为靶点的抗癌药物。
There was close relationship between protein acetylation and the tumor development. The state of Acetylation depended on the activity competetion between histone acetyltransferase (HATs) and histone deacetylase(HDACs).
Lots of experiences shows that HDACs inhibitor could increase the level of acetylation of histones in chromatin causing the activation and expression of specific genes, accordingly leading to the end differentiation and apoptosis of cancer cells.
Objective:To establish a rapid, relatively quantitative method for detecting acetylated proteins, and to explore its applications.
Method:Different number of Jurkat cells were treated with different concentration of histone deacetylase inhibitor Trichostatin A(TSA) at different time periods. After enrichment and purification with anti-acetylated lysine antibodies affinity chromatography column and then elution by acid, the levels of acetylated protein of Jurkat cells were tested by ELISA; The eluted components were identified by MALDI-TOF-TOF mass spectrometry. A preliminary study on the change of levels of acetylated proteins of Jurkat cells treated with3different traditional medicine monomer, including gallic acid、Emodin and mono acetylated Emodin A, was performed by the method.
Result:The relative amount of total acetylated protein of Jurkat cells reached the highest level after4×105number of cells treated with1μmol/L of TSA for24h.22acetylated proteins were identified by MALDI-TOF-TOF in eluent among them15were acetylated histones; Compared with negative control group(without drug), the degree of acetylation of protein of each treatment group was increased, and that of1.47μmol/L of gallic acid group was increased most remarkedly(P<0.01).
Conclusion:The method was initially established to enricr、purify and detect the total acetylated proteins in living cells. It might be a fast, simple way for screening anticancer drugs which targeted to deacetylase.
HDAC抑制剂会使染色质组蛋白乙酰化水平提高,因此导致特定基因激活表达,相应地导致细胞的末端分化或癌细胞的凋亡。初步的临床研究表明,人类可以安全地通过抑制HDAC的活性来获得组蛋白高乙酰化水平,HDACs已经成为抗癌新药开发的热门靶点,而研发这一类抗癌新药的前条件之一就是要建立适应于药物筛选所用快速、相对定量检测乙酰化蛋白方法。
目的：旨在建立一种快速、相对定量检测乙酰化蛋白方法,并初步探讨其应用。
方法：本文用乙酰化蛋白标准品寻找最优的质谱前处理方案,并将其应用于检测乙酰化蛋白方法的建模。模型建立如下：不同浓度的曲古菌素A(Trichostatin A.TSA)于不同的时间段刺激不同数量的Jurkat细胞,诱导其蛋白高乙酰化后,使用抗乙酰化赖氨酸抗体亲和层析柱富集纯化乙酰化总蛋白,经酸洗脱后固定于酶标板,ELISA检测乙酰化蛋白相对总量,并用MALDI-TOF-TOF分析验证其成分。然后利用该方法初步检测不同浓度的没食子酸、大黄素、单乙酰化大黄素A作用Jurkat细胞后乙酰化总蛋白水平的变化。
结果：1μmol/L的TSA作用于4×105Jurkat细胞24h,乙酰化总蛋白相对水平最高。MALDI-TOF-TOF分析显示,TSA诱导Jurkat细胞产生的乙酰化蛋白共有22种,其中15种为乙酰化组蛋白。
没食子酸、大黄素、单乙酰化大黄素A作用于Jurkat细胞后所导致的乙酰化程度不同,以1μmol/L浓度TSA为阳性对照组,无药物为空白对照组,35.09μmol/L和17.54μmol/L大黄素处理的Jurkat细胞蛋白乙酰化相对水平分别为4.3%和14.2%；1.47μmol/L和2.94μmol/L没食子酸处理组相对蛋白乙酰化水平分别为28.7%和11.5%；152.91μmol/L和30.58μmol/L单乙酰化大黄素组分别为22.0%和3.6%。其中1.47μmol/L没食子酸所诱导的乙酰化水平最高。
结论：初步建立了活细胞基础上纯化富集并检测乙酰化总蛋白水平的方法,该法可快速、简便的筛选以组蛋白去乙酰化酶为靶点的抗癌药物。
There was close relationship between protein acetylation and the tumor development. The state of Acetylation depended on the activity competetion between histone acetyltransferase (HATs) and histone deacetylase(HDACs).
Lots of experiences shows that HDACs inhibitor could increase the level of acetylation of histones in chromatin causing the activation and expression of specific genes, accordingly leading to the end differentiation and apoptosis of cancer cells.
Objective:To establish a rapid, relatively quantitative method for detecting acetylated proteins, and to explore its applications.
Method:Different number of Jurkat cells were treated with different concentration of histone deacetylase inhibitor Trichostatin A(TSA) at different time periods. After enrichment and purification with anti-acetylated lysine antibodies affinity chromatography column and then elution by acid, the levels of acetylated protein of Jurkat cells were tested by ELISA; The eluted components were identified by MALDI-TOF-TOF mass spectrometry. A preliminary study on the change of levels of acetylated proteins of Jurkat cells treated with3different traditional medicine monomer, including gallic acid、Emodin and mono acetylated Emodin A, was performed by the method.
Result:The relative amount of total acetylated protein of Jurkat cells reached the highest level after4×105number of cells treated with1μmol/L of TSA for24h.22acetylated proteins were identified by MALDI-TOF-TOF in eluent among them15were acetylated histones; Compared with negative control group(without drug), the degree of acetylation of protein of each treatment group was increased, and that of1.47μmol/L of gallic acid group was increased most remarkedly(P<0.01).
Conclusion:The method was initially established to enricr、purify and detect the total acetylated proteins in living cells. It might be a fast, simple way for screening anticancer drugs which targeted to deacetylase.
引文
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