High-Resolution Parallel Reaction Monitoring with Electron Transfer Dissociation for Middle-Down Proteomics: An Application to Study the Quantitative Changes Induced by Histone Modifying Enzyme Inhibitors and Activators

组蛋白 化学 乙酰化 组蛋白脱乙酰基酶 选择性反应监测 蛋白质组学 电子转移离解 定量蛋白质组学 生物化学 计算生物学 串联质谱法 组合化学 质谱法 色谱法 生物 DNA 基因
作者
Michael J. Sweredoski,Annie Moradian,Sonja Hess
出处
期刊:Methods in molecular biology [Springer Science+Business Media]
卷期号:: 61-69 被引量:5
标识
DOI:10.1007/978-1-4939-7201-2_4
摘要

With the advent of new methodologies, proteomics-based assays are increasingly used to study the efficacy of drugs on a molecular level. For these studies to be meaningful, the proteomics assays need to be sensitive, selective, accurate, and reproducible. This is often accomplished through a targeted approach, either using single or multiple reaction monitoring (SRM/MRM) or, more recently, parallel reaction monitoring (PRM). In PRM, the parallel detection of all product ions in a high-resolution mass spectrometer affords higher selectivity than SRM/MRM. PRM is thus better suited to analyze peptides larger than 2 kDa. Similar to SRM/MRM, PRM provides sensitive, accurate, and reproducible quantitative data. Here, we present a specific PRM method to characterize the effects of histone modifying enzyme drugs such as histone deacetylase inhibitors (HDAC) on the posttranslational modifications of histones, in a quantitative manner. More specifically, we characterize the heavily modified N-terminal tail of histone H3 after treatment with the HDAC inhibitor butyric acid, and monitor the acetylation and methylation events after treatment. To take most advantage of the multiply charged N-terminal histone peptides that are generated by an endoproteinase GluC-digestion, we use electron transfer dissociation (ETD) as the method of MS/MS fragmentation. This provides high sequence coverage for the modified peptides. The methodology is not limited to HDAC inhibitors, and can be used for any modifying enzyme. In fact, it can even be expanded beyond histone analyses. To give guidance for the development of a PRM assay, we present here HDAC inhibited H3 histone N-terminal tails as an example.
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