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.

Simultaneous Proteoform Analysis of Histones H3 and H4 with a Simplified Middle-Down Proteomics Method

“…ETD (compared with collision-induced dissociation (CID) or HCD) generates better sequence coverage for larger histone peptides and peptides with higher charge states. 36 The analysis shows good dispersion of multiple acetylation and methylation marks (Figure 3), showing a staggered elution of H3 and H4 modification states. The chromatography is reproducible as proven by technical replicates (not shown).…”

“…The injected sample amount was ∼0.1 μg, and ETD fragmentation was used in the data-dependent acquisition of MS/MS spectra. ETD (compared with collision-induced dissociation (CID) or HCD) generates better sequence coverage for larger histone peptides and peptides with higher charge states . The analysis shows good dispersion of multiple acetylation and methylation marks (Figure ), showing a staggered elution of H3 and H4 modification states.…”

Middle-down approach: a choice to sequence and characterize proteins/proteomes by mass spectrometry

“…64,67,68,77,79,85,91,139 Additionally, a few MD studies have been carried out using the hybrid LTQ-FT-ICR-MS also. 75,84,185,186…”

“…[76][77][78][79][80]82,173 Skyline soware also has been utilized for quantifying histones, which were characterized by MD approach. 186 Other soware, such as Pro-sightPC and MASH-Suite, which were primarily developed for analysis of TD proteomic data, have also been applied well for the purpose of analyzing MD proteomic data. 66,67,70,88,89,99,172,185 Algorithms to process the raw data of MDP for the purpose of deconvolution are, Xtract, THRASH 249 and MS-Deconv.…”

Computational Approaches in Proteomics

“…Since MD proteomics research has a considerable impact on PTM research, specific software tools have been created to analyze PTMs and relevant data. Among these tools are the previously mentioned isoScale software and the Skyline software(55). MD proteomics is still lacking established and standardized tools suitable for data interpretation, and although algorithms and software tools remain under constant development and improvement, this issue is mainly overcome by using TD proteomics tools instead.…”