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Author:
Date:
2020-09-20
[Abstract] Chromatin modifications, like histone post translational modifications (PTMs), are critical for tuning gene expression and many other aspects of cell phenotype. Liquid chromatography coupled to mass spectrometry (LC-MS) has become the most suitable method to analyze histones and histone PTMs in a large-scale manner. Selected histone PTMs have known functions, and their aberrant regulation is linked to a wide variety of diseases, including cancer. However, histone analysis is scarcely used in diagnostics, partially due to the limited throughput and not ideal reproducibility of LC-MS based analysis. We describe a workflow that allows for high-throughput sample preparation is less than a day using 96-well plates. Following preparation, samples are sprayed into MS without LC, using an ...
[摘要] [抽象]像组蛋白翻译后修饰（PTM）一样，染色质修饰对于调节基因表达和细胞表型的许多其他方面至关重要。液相色谱-质谱联用（LC-MS）已成为最适合大规模分析组蛋白和组蛋白PTM的方法。选定的组蛋白PTM具有已知功能，其异常调节与包括癌症在内的多种疾病有关。但是，组蛋白分析很少用于诊断中，部分是由于通量有限且基于LC-MS的分析的重现性不理想。我们描述了一种使用96孔板进行少于一天的高通量样品制备的工作流程。制备后，使用自动直接进样（DI-MS）方法将样品喷雾到无LC的MS中。每次分析都可以通过45个PTM（甲基化，乙酰化和磷酸化（共151个组蛋白标记）和16个未修饰的组蛋白肽进行组蛋白变体的相对定量。由于没有残留或基于LC的批处理效应，该工作流程允许MS运行少于1分钟，并具有更高的重现性和耐用性。最后，我们描述了一种工程化的肽序列，用于精确监控样品制备的效率，可以在DI-MS运行期间检测到该效率。
[背景] 组蛋白是具有球形头部和N末端尾巴的碱性蛋白质，富含精氨酸和赖氨酸残基。一对典型的组蛋白H2A，H2B，H3和H4（称为核心组蛋白）形成一个八聚体，其周围147 ...
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