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Top-Down Ion Mobility Separations of Isomeric Proteoforms.

Francis Berthias1, Hayden A Thurman2, Gayani Wijegunawardena2

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Researchers achieved baseline ion mobility spectrometry (IMS) resolution of intact isomeric proteoforms, specifically acetylated histone H4. This breakthrough enables detailed analysis of post-translational modification patterns crucial for epigenetics and proteomics.

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Area of Science:

  • Proteomics
  • Epigenetics
  • Biochemistry

Background:

  • Proteoforms, proteins with varied sequences or post-translational modifications (PTMs), are biologically crucial.
  • Histone PTM patterns regulate DNA transcription, but isomeric proteoforms often require separation for mass spectrometry (MS).
  • Current methods like chromatography or ion mobility spectrometry (IMS) can separate proteoforms but may lose PTM connectivity information.

Purpose of the Study:

  • To demonstrate baseline IMS resolution of intact isomeric proteoforms.
  • To separate and characterize acetylated histone H4 proteoforms with single acetylations on different lysine residues.
  • To establish a novel top-down proteoform characterization method.

Main Methods:

  • Synthesized specific acetylated histone H4 proteoforms using two-step native chemical ligation.
  • Employed trapped ion mobility spectrometry (TIMS) on a Bruker TIMS/ToF platform for separation.
  • Utilized tandem mass spectrometry (MS/MS) with collision-induced dissociation (CID) for confirmation.

Main Results:

  • Achieved baseline IMS resolution of intact isomeric acetylated histone H4 proteoforms (11.3 kDa).
  • Demonstrated separation of proteoforms with single acetylation on K5, K8, K12, K16, and K20.
  • Confirmed full resolution using binary mixtures and identified unique fragments via MS/MS.

Conclusions:

  • Established a novel capability for top-down proteoform characterization.
  • IMS resolution of intact isomeric proteoforms opens new avenues in proteomics and epigenetics.
  • This method preserves crucial PTM connectivity information.