Abstract
Histones represent a class of proteins ideally suited to analyses by top-down mass spectrometry due to their relatively small size, the high electron transfer dissociation-compatible charge states they exhibit, and the potential to gain valuable information concerning combinatorial post-translational modifications and variants. We recently described new methods in mass spectrometry for the acquisition of high-quality MS/MS spectra of intact proteins (Anderson, L. C., English, A. M., Wang, W., Bai, D. L., Shabanowitz, J., and Hunt, D. F. (2015) Int. J. Mass Spectrom. 377, 617-624). Here, we report an extension of these techniques. Sequential ion/ion reactions carried out in a modified Orbitrap Velos Pro/Elite(TM) capable of multiple fragment ion fills of the C-trap, in combination with data-dependent and targeted HPLC-MS experiments, were used to obtain high resolution MS/MS spectra of histones from butyrate-treated HeLa cells. These spectra were used to identify several unique intact histone proteoforms with up to 81% sequence coverage. We also demonstrate that parallel ion parking during ion/ion proton transfer reactions can be used to separate species of overlapping m/z that are not separated chromatographically, revealing previously indiscernible signals. Finally, we characterized several truncated forms of H2A and H2B found within the histone fractions analyzed, achieving up to 93% sequence coverage by electron transfer dissociation MS/MS. Results of follow-up in vitro experiments suggest that some of the truncated histone H2A proteoforms we observed can be generated by cathepsin L, an enzyme known to also catalyze clipping of histone H3.
Highlights
Chromatin is the structural framework that packages DNA into chromosomes within the nucleus of a cell [2]
In a 2014 Proteomics article authored by members of the Consortium for Top-Down Proteomics, Dang et al concluded that analyses of intact proteins could be greatly improved by the development of higher resolution isolation capabilities and by the ability to acquire rich sequence informative mass spectrometry (MS)/MS spectra on a time scale more compatible with chromatography [18]
Once sufficient amounts of fragment ions are collected, they are sent to the Orbitrap for high resolution mass analysis
Summary
Analyses of Histone Proteoforms Using Frontend Electron Transfer Dissociation-enabled Orbitrap Instruments*□S. Sequential ion/ion reactions carried out in a modified Orbitrap Velos Pro/EliteTM capable of multiple fragment ion fills of the C-trap, in combination with datadependent and targeted HPLC-MS experiments, were used to obtain high resolution MS/MS spectra of histones from butyrate-treated HeLa cells These spectra were used to identify several unique intact histone proteoforms with up to 81% sequence coverage. With the advent of electron transfer dissociation (ETD) and more efficient electron capture dissociation fragmentation methods, which are better suited for larger, more highly charged peptides [12, 13], several studies utilizing other endoproteases to generate longer peptides have emerged (14 – 16) These methodologies do well to preserve the combinatorial PTM profiles of histone tails, in some cases it is still impossible to identify the proteoforms from which these peptides originate. We demonstrate the power of these new techniques through their coupling with on-line, nano-LC chromatography and application to the analysis of intact histones derived from butyrate-treated HeLa cells
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