Abstract
The use of electron-transfer dissociation as an alternative peptide ion activation method for generation of protein sequence information is examined here in comparison with the conventional method of choice, collisionally activated dissociation, using a linear ion trapping instrument. Direct comparability between collisionally and electron-transfer-activated product ion data were ensured by employing an activation-switching method during acquisition, sequentially activating precisely the same precursor ion species with each fragmentation method in turn. Sequest (Thermo Fisher Scientific, San Jose, CA) searching of product ion data generated an overlapping yet distinct pool of polypeptide identifications from the products of collisional and electron-transfer-mediated activation products. To provide a highly confident set of protein recognitions, identification data were filtered using parameters that achieved a peptide false discovery rate of 1%, with two or more independent peptide assignments required for each protein. The use of electron transfer dissociation (ETD) has allowed us to identify additional peptides where the quality of product ion data generated by collisionally activated dissociation (CAD) was insufficient to infer peptide sequence. Thus, a combined ETD/CAD approach leads to the recognition of more peptides and proteins than are achieved using peptide analysis by CAD- or ETD-based tandem mass spectrometry alone.
Article PDF
Similar content being viewed by others
References
Simpson, R. J.; Connolly, L. M.; Eddes, J. S.; Pereira, J. J.; Moritz, R. L.; Reid, G. E. Proteomic Analysis of the Human Colon Carcinoma Cell Line (LIM 1215): Development of a Membrane Protein Database. Electrophoresis 2000, 21, 1707–1732.
Steen, H.; Mann, M. The ABC’s (and XYZ’s) of Peptide Sequencing. Nat. Rev. Mol. Cell Biol. 2004, 5, 699–711.
Aebersold, R.; Goodlett, D. R. Mass Spectrometry in Proteomics. Chem. Rev. 2001, 101, 269–295.
Larsen, M. R.; Trelle, M. B.; Thingholm, T. E.; Jensen, O. N. Analysis of Post-Translational Modifications of Proteins by Tandem Mass Spectrometry. Biotechniques 2006, 40, 790–798.
Smith, R. D.; Barinaga, C. J.; Udseth, H. R. Tandem Mass-Spectrometry of Highly Charged Cytochrome c Molecular-Ions Produced by Electrospray Ionization. J. Phys. Chem. 1989, 93, 5019–5022.
Nemeth-Cawley, J. F.; Rouse, J. C. Identification and Sequencing Analysis of Intact Proteins Via Collision-Induced Dissociation and Quadrupole Time-of-Flight Mass Spectrometry. J. Mass Spectrom. 2002, 37, 270–282.
Raspopov, S. A.; El-Faramawy, A.; Thomson, B. A.; Siu, K. W. Infrared Multiphoton Dissociation in Quadrupole Time-of-Flight Mass Spectrometry: Top-Down Characterization of Proteins. Anal. Chem. 2006, 78, 4572–4577.
Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. Electron Capture Dissociation of Multiply Charged Protein Cations: A Nonergodic Process. J. Am. Chem. Soc. 1998, 120, 3265–3266.
Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N. A.; Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W. Electron Capture Dissociation for Structural Characterization of Multiply Charged Protein Cations. Anal. Chem. 2000, 72, 563–573.
Breuker, K.; Oh, H.; Lin, C.; Carpenter, B. K.; McLafferty, F. W. Nonergodic and Conformational Control of the Electron Capture Dissociation of Protein Cations. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14011–14016.
Syrstad, E. A.; Turecek, F. Toward a General Mechanism of Electron Capture Dissociation. J. Am. Soc. Mass Spectrom. 2005, 16, 208–224.
Hudgins, R. R.; Håkansson, K.; Quinn, J. P.; Hendrickson, C. L.; Marshall, A. G., 50th American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics, May 2002, Orlando, FL.
Zubarev, R. A. Reactions of Polypeptide Ions with Electrons in the Gas Phase. Mass Spectrom. Rev. 2003, 22, 57–77.
Chalmers, M. J.; Hakansson, K.; Johnson, R.; Smith, R.; Shen, J.; Emmett, M. R.; Marshall, A. G. Protein Kinase A Phosphorylation Characterized by Tandem Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Proteomics 2004, 4, 970–981.
Cooper, H. J.; Hakansson, K.; Marshall, A. G. The Role of Electron Capture Dissociation in Biomolecular Analysis. Mass Spectrom. Rev. 2005, 24, 201–222.
Creese, A. J.; Cooper, H. J. Liquid Chromatography Electron Capture Dissociation Tandem Mass Spectrometry (LC-ECD-MS/MS) Versus Liquid Chromatography Collision-Induced Dissociation Tandem Mass Spectrometry (LC-CID-MS/MS) for the Identification of Proteins. J. Am. Soc. Mass Spectrom. 2007, 18, 891–897.
Syka, J. E.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Peptide and Protein Sequence Analysis by Electron Transfer Dissociation Mass Spectrometry. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9528–9533.
Brekenfeld, A.; Ledertheil, T.; Lubeck, M.; Baessmann, C.; Hartmer, R., 53rd American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics, May 2005, San Antonio, TX.
Coon, J. J.; Ueberheide, B.; Syka, J. E.; Dryhurst, D. D.; Ausio, J.; Shabanowitz, J.; Hunt, D. F. Protein Identification Using Sequential Ion/Ion Reactions and Tandem Mass Spectrometry. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9463–9468.
Good, D. M.; Wirtala, M.; McAlister, G. C.; Coon, J. J. Performance Characteristics of Electron Transfer Dissociation Mass Spectrometry. Mol. Cell. Proteom. 2007, 6, 1942–1951.
Broadhead, R.; Dawe, H. R.; Farr, H.; Griffiths, S.; Hart, S. R.; Portman, N.; Shaw, M. K.; Ginger, M. L.; Gaskell, S. J.; McKean, P. G.; Gull, K. Flagellar Motility is Required for the Viability of the Bloodstream Trypanosome. Nature 2006, 440, 224–227.
Peng, J.; Elias, J. E.; Thoreen, C. C.; Licklider, L. J.; Gygi, S. P. Evaluation of Multidimensional Chromatography Coupled with Tandem Mass Spectrometry (LC/LC-MS/MS) for Large-Scale Protein Analysis: The Yeast Proteome. J. Proteome Res. 2003, 2, 43–50.
Lundgren, D. H.; Han, D. K.; Eng, J. K. Protein Identification using TurboSEQUEST. Curr. Protoc. Bioinformatics. 2005, Chap. 13, Unit 13.3.
Good, D. M.; Coon, J. J. Advancing Proteomics with ion/ion Chemistry. Biotechniques 2006, 40, 783–789.
Elias, J. E.; Gygi, S. P. Target-Decoy Search Strategy for Increased Confidence in Large-Scale Protein Identifications by Mass Spectrometry. Nat. Methods 2007, 4, 207–214.
Swaney, D. L.; McAlister, G. C.; Wirtala, M.; Schwartz, J. C.; Syka, J. E.; Coon, J. J. Supplemental Activation Method for High-Efficiency Electron-Transfer Dissociation of Doubly Protonated Peptide Precursors. Anal. Chem. 2007, 79, 477–485.
Molina, H.; Matthiesen, R.; Kandasamy, K.; Pandey, A. Comprehensive Comparison of Collision Induced Dissociation and Electron Transfer Dissociation. Anal. Chem. 2008, 80, 4825–4835.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published online September 3, 2008
Rights and permissions
About this article
Cite this article
Hart, S.R., Lau, K.W., Hao, Z. et al. Analysis of the trypanosome flagellar proteome using a combined electron transfer/collisionally activated dissociation strategy. J Am Soc Mass Spectrom 20, 167–175 (2009). https://doi.org/10.1016/j.jasms.2008.08.014
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1016/j.jasms.2008.08.014