One minute analysis of 200 histone posttranslational modifications by direct injection mass spectrometry
One minute analysis of 200 histone posttranslational modifications by direct injection mass spectrometry
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DOI：
10.1101/gr.247353.118
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发表时间：
2019-06-01
期刊：
影响因子：
7
通讯作者：
Garcia, Benjamin A.
中科院分区：
文献类型：
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作者：
Sidoli, Simone;Kori, Yekaterina;Garcia, Benjamin A.
DNA 和组蛋白定义了染色质的结构和组成。组蛋白翻译后修饰 (PTM) 是能够模拟染色质可及性的共价化学基团，主要是因为它们能够招募负责 DNA 读出和重塑的酶。基于质谱 (MS) 的蛋白质组学是大规模鉴定和定量蛋白质 PTM（包括组蛋白）的首选方法。高灵敏度蛋白质组学需要在线 MS 耦合，通量相对较低，纳米液相色谱 (nanoLC) 稳健性较差，对于组蛋白，需要进行包括组蛋白纯化、衍生化和消化在内的二维样品制备。我们提出了一种新方案，可在 7 小时内从开始到结束获得来自组织或细胞系的约 200 个组蛋白 PTM 的定量数据。该方案包括 4 小时的组蛋白提取、3 小时的衍生化和消化，以及仅 1 分钟的通过直接注射 (DI-MS) 的 MS 分析。我们证明，通过使用多通道移液器和 96 孔板，可以并行处理 384 个样品。我们还设计了合成“组蛋白样”肽的序列以掺入样品中，其衍生化和消化作为样品制备质量的基准。通过生成和分析等摩尔混合的合成修饰组蛋白肽文库，我们确保 DI-MS 与 nanoLC-MS/MS 相比不会在组蛋白肽电离中引入偏差。最后，我们引入 EpiProfileLite 来全面分析这种新数据类型。总而言之，我们的工作流程适合使用单个质谱仪每天对 > 1000 个样品进行高通量筛选。
DNA and histone proteins define the structure and composition of chromatin. Histone posttranslational modifications (PTMs) are covalent chemical groups capable of modeling chromatin accessibility, mostly due to their ability in recruiting enzymes responsible for DNA readout and remodeling. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein PTMs, including histones. High sensitivity proteomics requires online MS coupling with relatively low throughput and poorly robust nano-liquid chromatography (nanoLC) and, for histone proteins, a 2-d sample preparation that includes histone purification, derivatization, and digestion. We present a new protocol that achieves quantitative data on about 200 histone PTMs from tissue or cell lines in 7 h from start to finish. This protocol includes 4 h of histone extraction, 3 h of derivatization and digestion, and only 1 min of MS analysis via direct injection (DI-MS). We demonstrate that this sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates. We also engineered the sequence of a synthetic "histone-like" peptide to spike into the sample, of which derivatization and digestion benchmarks the quality of the sample preparation. We ensure that DI-MS does not introduce biases in histone peptide ionization as compared to nanoLC-MS/MS by producing and analyzing a library of synthetically modified histone peptides mixed in equal molarity. Finally, we introduce EpiProfileLite for comprehensive analysis of this new data type. Altogether, our workflow is suitable for high-throughput screening of >1000 samples per day using a single mass spectrometer.