Bhanu Natarajan V, Sidoli Simone, Garcia Benjamin A
Epigenetics Institute, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Bio Protoc. 2020 Sep 20;10(18):e3756. doi: 10.21769/BioProtoc.3756.
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 automated direct injection (DI-MS) method. Each analysis provides accurate quantification for 29 peptide sequences with 45 PTMs (methylations, acetylations and phosphorylations) for a total of 151 histone marks plus 16 unmodified histone peptides for relative quantification of histone variants. This workflow allows for < 1 min MS runs and higher reproducibility and robustness due to the absence of carryover or LC-based batch effects. Finally, we describe an engineered peptide sequence used to accurately monitor the efficiency of sample preparation, which can be detected during the DI-MS run.
染色质修饰，如组蛋白翻译后修饰（PTMs），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）已成为大规模分析组蛋白和组蛋白PTMs的最合适方法。特定的组蛋白PTMs具有已知功能，其异常调控与包括癌症在内的多种疾病相关。然而，组蛋白分析在诊断中很少使用，部分原因是基于LC-MS的分析通量有限且重现性不理想。我们描述了一种工作流程，使用96孔板可在不到一天的时间内实现高通量样品制备。制备后，使用自动直接进样（DI-MS）方法将样品不经LC直接喷入质谱仪。每次分析可为29个肽序列提供准确的定量，这些肽序列带有45种PTMs（甲基化、乙酰化和磷酸化），总共151个组蛋白标记，外加16个未修饰的组蛋白肽用于组蛋白变体的相对定量。由于不存在残留或基于LC的批次效应，该工作流程允许质谱运行时间<1分钟，且具有更高的重现性和稳健性。最后，我们描述了一种用于准确监测样品制备效率的工程化肽序列，该序列可在DI-MS运行期间检测到。
