Quantitative Assessment of Chromatin Immunoprecipitation Grade Antibodies Directed against Histone Modifications Reveals Patterns of Co-occurring Marks on Histone Protein Molecules
Quantitative Assessment of Chromatin Immunoprecipitation Grade Antibodies Directed against Histone Modifications Reveals Patterns of Co-occurring Marks on Histone Protein Molecules
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DOI：
10.1074/mcp.m111.015941
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发表时间：
2012-05-01
影响因子：
7
通讯作者：
Jaffe, Jacob D.
中科院分区：
文献类型：
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作者：
Peach, Sally E.;Rudomin, Emily L.;Jaffe, Jacob D.
在大多数染色质免疫沉淀(CHIP)分析中，确定步骤是使用抗体来丰富与染色质片段相关的特定蛋白质或组蛋白修饰状态。抗体的特异性是解释实验的关键，但这种特性很少有报道。在这里，我们提出了一种使用质谱学的定量方法来表征关键的组蛋白H3修饰的特异性-靶向抗体，这些抗体以前被用来表征“组蛋白密码”。我们进一步扩展了这些抗体试剂的使用，以观察不同的组蛋白修饰之间的长期相关性。使用代表活细胞中染色质状态混合物的纯化的人组蛋白，我们能够量化靶向浓缩的程度和几种常用的、商业上可用的芯片级抗体的特异性。针对H3K4me2、H3K4me3、H3K9me3和H3K27me3四种常见的染色质标记，我们发现不同试剂的浓缩效率存在显著差异。对于一些抗体，我们还检测到在同一位置有显著的非靶标交替修饰浓缩(即，通过针对H3K4me3的抗体浓缩H3K4me2)。通过聚类分析，我们能够识别同一组蛋白上不同位置标记的共浓缩模式。令人惊讶的是，这些共富集很好地对应于染色质的典型状态，即染色质的激活和抑制区域。总之，我们的发现表明，1)考虑到常用的组蛋白修饰识别抗体的交叉反应的可能性，我们需要谨慎地评估CHIP实验的结果，2)同一组蛋白分子上存在多个具有一致生物学解释的标记，以及3)组蛋白密码的某些成分可能被转导到活细胞中的单个蛋白质上。分子与细胞蛋白质组学11：10.1074/mcp.M111.015941,128-137,2012年。
The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.015941, 128-137, 2012.