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

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.