title: Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments.
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.
authors.affiliation: From the ‡Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904;
authors.forename: Lissa C
authors.initials: LC
authors.lastname: Anderson
authors.affiliation: the §Epigenetics Program and Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and.
authors.forename: Kelly R
authors.initials: KR
authors.lastname: Karch
authors.affiliation: From the ‡Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904;
authors.forename: Scott A
authors.initials: SA
authors.lastname: Ugrin
authors.affiliation: the §Epigenetics Program and Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and.
authors.forename: Mariel
authors.initials: M
authors.lastname: Coradin
authors.affiliation: From the ‡Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904;
authors.forename: A Michelle
authors.initials: AM
authors.lastname: English
authors.affiliation: the §Epigenetics Program and Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and.
authors.forename: Simone
authors.initials: S
authors.lastname: Sidoli
authors.affiliation: From the ‡Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904;
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shabanowitz
authors.affiliation: the §Epigenetics Program and Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Garcia
authors.affiliation: From the ‡Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904; the ¶Department of Pathology, University of Virginia, Charlottesville, Virginia 22908 dfh@virginia.edu.
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
mesh_terms: D002087:Butyrates / Q000737:chemistry; D014644:Genetic Variation; D006367:HeLa Cells; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation*
chemical_list: D002087:Butyrates; D006657:Histones; D020543:Proteome