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

Annie Moradian,Sonja Hess,Catarina Franco,Michael J Sweredoski

doi:10.1186/s40543-015-0060-7

Abstract

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.

Highlights

The post-translational modifications (PTMs) of histones play a major role in activating or silencing gene transcription
The use of weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC) enabled the separation of histones based on their charge and hydrophilic interactions (Young et al 2009; Young et al 2010)
While the peaks may appear relatively broad in comparison to C18 reversed-phase chromatography, WCX-HILIC affords a secondary dimension of separation based on the methylation status (Fig. 3b), separating multiple methylated isoforms that contain the same number of acetylations

Summary

Introduction

The post-translational modifications (PTMs) of histones play a major role in activating or silencing gene transcription. * Correspondence: shess@caltech.edu 1Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, BI 211, MC 139-74, Pasadena, CA 91125, USA Full list of author information is available at the end of the article amine of the lysine by forming an amide bond (Fig. 1a). Acetylated and neutral lysines cannot interact closely with DNA, leading to an open chromatin structure, which is commonly associated with transcriptional activation (Fig. 1b) (Garcia et al 2007).

Results

Conclusion