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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

Sweredoski, Michael J. and Moradian, Annie and Hess, Sonja (2017) 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. In: Proteomics for Drug Discovery. Methods in Molecular Biology. No.1647. Humana Press , New York, NY, pp. 61-69. ISBN 978-1-4939-7200-5. https://resolver.caltech.edu/CaltechAUTHORS:20170817-081105012

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Abstract

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.


Item Type:Book Section
Related URLs:
URLURL TypeDescription
https://doi.org/10.1007/978-1-4939-7201-2_4DOIArticle
https://link.springer.com/protocol/10.1007%2F978-1-4939-7201-2_4PublisherArticle
ORCID:
AuthorORCID
Sweredoski, Michael J.0000-0003-0878-3831
Moradian, Annie0000-0002-0407-2031
Hess, Sonja0000-0002-5904-9816
Additional Information:© 2017 Springer Science+Business Media LLC. First Online: 15 August 2017.
Subject Keywords:Parallel reaction monitoring (PRM); Quadrupole-Orbitrap mass spectrometry; Quantitation; Histones; Electron transfer dissociation (ETD)
Series Name:Methods in Molecular Biology
Issue or Number:1647
Record Number:CaltechAUTHORS:20170817-081105012
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170817-081105012
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:80549
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:17 Aug 2017 16:02
Last Modified:22 Nov 2019 09:58

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