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Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures
Published on: September 28, 2013
Benchmarking multiple fragmentation methods on an orbitrap fusion for top-down phospho-proteoform characterization.
Andrea M Brunner1,2, Philip Lössl1,2, Fan Liu1,2
1†Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584CH Utrecht, The Netherlands.
Top-down mass spectrometry improves proteoform characterization using the novel EThcD fragmentation method. This technique enhances protein sequence coverage and phosphosite localization for detailed analysis of post-translational modifications.
Area of Science:
- Proteomics
- Mass Spectrometry
- Molecular Biology
Background:
- Top-down mass spectrometry enables comprehensive proteoform characterization, including identifying co-occurring post-translational modifications (PTMs).
- Insufficient protein sequence coverage from incomplete fragmentation is a key limitation in top-down proteomics.
Purpose of the Study:
- To evaluate the efficacy of the dual fragmentation method, EThcD, for improving protein sequence coverage and phospho-proteoform characterization in intact protein analysis.
- To benchmark EThcD against other fragmentation methods on the Orbitrap Fusion platform.
Main Methods:
- Systematic benchmarking of fragmentation methods (EThcD, ETciD, HCD, ETD) for intact protein analysis.
- Utilized a 17.5 kDa N-terminal fragment of the mitotic regulator Bora as a model system.
- Monitored phosphorylation of Bora by Aurora A and Plk1 using top-down fragmentation.
Main Results:
- EThcD and ETciD methods are feasible on the Orbitrap Fusion, yielding richer fragmentation spectra than HCD or ETD alone.
- Demonstrated increased protein sequence coverage using EThcD and ETciD.
- Facilitated enhanced phosphosite localization and identification of kinase-specific phosphorylation sites.
Conclusions:
- The EThcD fragmentation method significantly improves protein sequence coverage in top-down mass spectrometry.
- EThcD enhances the characterization of phospho-proteoforms, aiding in the localization of post-translational modifications and determination of kinase-specific phosphorylation sites.
- This advancement offers a more comprehensive approach to analyzing complex protein modifications.
