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Published June 21, 2015 | Published
Journal Article Open

Middle-down electron capture dissociation and electron transfer dissociation for histone analysis

Abstract

The post-translational modifications (PTMs) of histones play a major role in activating or silencing gene transcription. To gain better understanding of the interplay between the PTMs that occur on histones, they are extensively studied using mass spectrometry techniques. Due to the abundance of lysines and arginines, the typical trypsin digestion has been found less favorable and GluC-digests have been explored as an alternative to yield larger peptides amenable to middle-down approaches. In addition, the use of weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC) and the use of electron-based fragmentation techniques were found to be advantageous for the in-depth characterization of histone variants containing multiple PTMs. As a test model, we used histones from MEL (murine erythroleukemia) cells treated with butyric acid or DMSO. After acid extraction, histone pellets were dried and fractionated using a reversed-phase C3 column. For middle-down analysis, selected histone fractions were digested using GluC. The digested samples were separated on a WCX-HILIC capillary column packed in-house with PolyCAT A resin, coupled to a linear trap quadrupole Fourier transformation ion cyclotron resonance (LTQFT-ICR) instrument. Raw data was acquired on the LTQFT-ICR using electron capture dissociation (ECD). After deconvolution of the raw data, we generated heatmaps to illustrate differential maps between differentially treated histone samples. We also explored the innovative use of Skyline to quantify histone tails. In addition, we report some preliminary data using a synthetic histone peptide acquired on an Orbitrap Fusion using electron transfer dissociation (ETD). Both, ECD and ETD methods are capable of comprehensively analyzing complex histone variations not accessible with conventional techniques.

Additional Information

© 2015 Moradian et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Received: 9 March 2015; Accepted: 19 May 2015; Published: 21 June 2015. The Proteome Exploration Laboratory is supported by the Gordon and Betty Moore Foundation through grant GBMF775 and the Beckman Institute; CF is supported by Fulbright, PROLAB (ASBMB) through a Wood-Whelan fellowship from IUBMB and from Fundação para a Ciência e Tecnologia (Lisbon, Portugal) with both a project grant (PTDC/MAR-BIO/2174/2012) and a post-doctoral fellowship (SFRH/BPD/79271/2011). The authors declare that they have no competing interests. Authors' contributions: AM and CF made the ECD and ETD measurements and wrote the article. MJS created the bioinformatics tools and wrote the article. SH conceived the project and wrote the review article. All authors read and approved the final manuscript.

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