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Enhanced Top-Down Protein Characterization with Electron Capture Dissociation and Cyclic Ion Mobility Spectrometry.

Jared B Shaw1, Dale A Cooper-Shepherd2, Darren Hewitt2

  • 1e-MSion Inc., 2121 NE Jack London Street, Corvallis, Oregon 97330, United States.

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Electron capture dissociation (ECD) combined with cyclic ion mobility spectrometry (cIMS) significantly improves top-down protein analysis. This method reduces spectral complexity, enabling higher protein sequence coverage, especially for large, highly charged proteins.

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Tandem mass spectrometry of large proteins produces complex spectra with overlapping signals.
  • Existing methods to reduce spectral congestion, such as improved mass analyzers and ion manipulation, have limitations.

Purpose of the Study:

  • To evaluate the utility of electron capture dissociation (ECD) coupled with high-resolution cyclic ion mobility spectrometry (cIMS) for enhanced top-down protein characterization.
  • To assess the impact of cIMS on reducing spectral congestion in ECD mass spectra.

Main Methods:

  • Utilized ECD for protein fragmentation.
  • Employed high-resolution cyclic ion mobility spectrometry (cIMS) to separate ECD product ions based on their drift time.
  • Analyzed "mobility fractions" (extracted mass spectra from specific drift time ranges) to decongest spectra.

Main Results:

  • For small proteins (e.g., ubiquitin), cIMS showed a modest increase in identified product ions but not sequence coverage.
  • For larger proteins (e.g., carbonic anhydrase), pre-IMS ECD combined with mobility fractions dramatically increased annotated isotope clusters (135%) and unique product ions (75%).
  • Achieved 89% sequence coverage for carbonic anhydrase using this combined approach.

Conclusions:

  • ECD coupled with cIMS significantly enhances top-down proteomics by reducing spectral complexity.
  • This approach is particularly beneficial for characterizing large and highly charged protein ions.
  • The combination of ECD and cIMS offers a powerful strategy for improving protein sequence coverage in mass spectrometry-based proteomics.