TY - JOUR
T1 - Analyses of histone proteoforms using frontend electron transfer dissociation-enabled orbitrap instruments
AU - Anderson, Lissa C.
AU - Karch, Kelly R.
AU - Ugrin, Scott A.
AU - Coradin, Mariel
AU - English, A. Michelle
AU - Sidoli, Simone
AU - Shabanowitz, Jeffrey
AU - Garcia, Benjamin A.
AU - Hunt, Donald F.
N1 - Funding Information:
We thank John E. P. Syka, Christopher Mullen, and Lee Early of Thermo Fisher Scientific for aiding in the modification of the Orbitrap Velos Pro to include FETD and for helpful discussion. We also thank Dina L. Bai for critical reading of early manuscript revisions and Greg T. Blakney for helpful discussion. This work was supported in part by National Institutes of Health Grants GM037537 and AI033993 (to D.F.H.). This work was also supported by NIH Grants GM110174 and AI118891 and a National Science Foundation Faculty Early Career Award (to B.A.G). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2016/3
Y1 - 2016/3
N2 - 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.
AB - 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.
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U2 - 10.1074/mcp.O115.053843
DO - 10.1074/mcp.O115.053843
M3 - Article
C2 - 26785730
AN - SCOPUS:84962562184
VL - 15
SP - 975
EP - 988
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
SN - 1535-9476
IS - 3
ER -