ETD Outperforms CID and HCD in the Analysis of the Ubiquitylated Proteome
Abstract
Comprehensive analysis of the ubiquitylome is a prerequisite to fully understand the regulatory role of ubiquitylation. However, the impact of key mass spectrometry parameters on ubiquitylome analyses has not been fully explored. In this study, we show that using electron transfer dissociation (ETD) fragmentation, either exclusively or as part of a decision tree method, leads to ca. 2-fold increase in ubiquitylation site identifications in K-ε-GG peptide-enriched samples over traditional collisional-induced dissociation (CID) or higher-energy collision dissociation (HCD) methods. Precursor ions were predominantly observed as 3+ charged species or higher and in a mass range 300–1200 m/z. N-ethylmaleimide was used as an alkylating agent to reduce false positive identifications resulting from overalkylation with halo-acetamides. These results demonstrate that the application of ETD fragmentation, in addition to narrowing the mass range and using N-ethylmaleimide yields more high-confidence ubiquitylation site identification than conventional CID and HCD analysis.
Additional Information
© 2015 American Society for Mass Spectrometry. Received: 12 February 2015; Revised: 3 April 2015; Accepted: 6 April 2015. The authors thank Dr. Raymond J. Deshaies for helpful suggestions and critical discussions of the work described. In addition, they thank Dr. Deshaies for kindly providing them with the yeast strain RJD 360 (W303 background). Lastly, they thank members of the Proteome Exploration Laboratory, housed in the Beckman Institute at the California Institute of Technology, for helpful discussions during the course of this work. This work was supported by the Gordon and Betty Moore Foundation, through grant GBMF775, the Beckman Institute and the NIH through grant 1S10RR029594-01A1.Attached Files
Accepted Version - nihms747417.pdf
Supplemental Material - ESM_1.pdf
Supplemental Material - Fig1.pdf
Supplemental Material - Table1.xlsx
Supplemental Material - Table2.xlsx
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Additional details
- PMCID
- PMC4711353
- Eprint ID
- 57946
- Resolver ID
- CaltechAUTHORS:20150602-145245304
- GBMF775
- Gordon and Betty Moore Foundation
- Caltech Beckman Institute
- 1S10RR029594-01A1
- NIH
- Created
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2015-06-02Created from EPrint's datestamp field
- Updated
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2022-06-03Created from EPrint's last_modified field