Middle-down electron capture dissociation and electron transfer dissociation for histone analysis
© Moradian et al. 2015
Received: 9 March 2015
Accepted: 19 May 2015
Published: 21 June 2015
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.
KeywordsHistone Post-translational modification Middle-down proteomics Electron capture dissociation Electron transfer dissociation Data-dependent analysis Data-independent analysis
In addition to ECD analyses on the LTQFT, we have started to explore suitable instrument parameters for ETD on an Orbitrap Fusion. To do so, preliminary data of the synthetic H3 histone N-terminal fragment ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALRE were acquired on an Orbitrap Fusion instrument.
Taken together, the use of WCX-HILIC with middle-down proteomics is becoming an increasingly popular method for histone characterization. Our preliminary data furthermore show that both the LTQFT with ECD acquisition and the Orbitrap Fusion with ETD acquisition are capable of identifying and quantifying histone PTMs. We are predicting that the Orbitrap Fusion will play an important role in the comprehensive and reproducible analysis of histones. Fully automated bioinformatics pipelines as they exist for bottom-up proteomics are not yet available for middle-down proteomics and will require substantial development. Once in place, they will likely trigger another significant advancement in histone modification analysis.
Electron capture dissociation
Electron transfer dissociation
Higher energy collision dissociation
Fourier transformation ion cyclotron resonance
Weak cation exchange hydrophilic interaction liquid chromatography
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).
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