Analyses of histone proteoforms using frontend electron transfer dissociation-enabled orbitrap instruments

Lissa C. Anderson, Kelly R. Karch, Scott A. Ugrin, Mariel Coradin, A. Michelle English, Simone Sidoli, Jeffrey Shabanowitz, Benjamin A. Garcia, Donald F. Hunt

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41 Scopus citations


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 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. 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.

Original languageEnglish (US)
Pages (from-to)975-988
Number of pages14
JournalMolecular and Cellular Proteomics
Issue number3
StatePublished - Mar 2016
Externally publishedYes

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology


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