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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.

SUBMITTER: Anderson LC

PROVIDER: S-EPMC4813714 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments.

Anderson Lissa C LC Karch Kelly R KR Ugrin Scott A SA Coradin Mariel M English A Michelle AM Sidoli Simone S Shabanowitz Jeffrey J Garcia Benjamin A BA Hunt Donald F DF

Molecular & cellular proteomics : MCP 20160119 3

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., Ba ...[more]

PMID: 26785730

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Project description:The increasing importance of immunoglobulins G (IgGs) as biotherapeutics calls for improved structural characterization methods designed for these large (~150kDa) macromolecules. Analysis workflows have to be rapid, robust, and require minimal sample preparation. In a previous work we showed the potential of Orbitrap Fourier transform mass spectrometry (FTMS) combined with electron transfer dissociation (ETD) for the top-down investigation of an intact IgG1, resulting in ~30% sequence coverage. Here, we describe a top-down analysis of two IgGs1 (adalimumab and trastuzumab) and one IgG2 (panitumumab) performed with ETD on a mass spectrometer equipped with a high-field Orbitrap mass analyzer. For the IgGs1, sequence coverage comparable to the previous results was achieved in a two-fold reduced number of summed transients, which corresponds, taken together with the significantly increased spectra acquisition rate, to ~six-fold improvement in analysis time. Furthermore, we studied the influence of ion-ion interaction times on ETD product ions for IgGs1, and the differences in fragmentation behavior between IgGs1 and IgG2, which present structural differences. Overall, these results reinforce the hypothesis that gas phase dissociation using both energy threshold-based and radical-driven ion activations is directed to specific regions of the polypeptide chains mostly by the location of disulfide bonds.Significance of the studyCompared with our previous report, the results presented herein demonstrate the power of technological advances of the next generation Orbitrap™ platform, including the use of a high-field compact (i.e., D20) Orbitrap mass analyzer, and a dedicated manipulation strategy for large protein ions (via their trapping in the HCD collision cell along with reduction of the pressure in the cell). Notably, these important developments became recently commercially available in the top-end Orbitrap platforms under the name of "Protein Mode". Furthermore, we continued exploring the advantages offered by the summation (averaging) of transients (time-domain data) for improving the signal-to-noise ratio of top-down mass spectra. Finally, for the first time we report the application of the hybrid ion activation technique that combines electron transfer dissociation and higher energy collisional dissociation, known as EThcD, on intact monoclonal antibodies. Under these specific instrumental parameters, EThcD produces a partially complementary fragmentation pattern compared to ETD, increasing the overall sequence coverage especially at the protein termini.

Dataset Information

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments.

Publications

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments.

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