Tandem mass spectrometry investigation of ADP-ribosylated kemptide

  • Shawna M. Hengel
  • Scott A. Shaffer
  • Brook L. Nunn
  • David R. Goodlett


Bacterial adenosine diphosphate-ribosyltransferases (ADPRTs) are toxins that play a significant role in pathogenicity by inactivating host proteins through covalent addition of ADP-ribose. In this study we used ADP-ribosylated Kemptide (LRRASLG) as a standard to examine the effectiveness of three common tandem mass spectrometry fragmentation methods for assignment of amino acid sequence and site of modification. Fragmentation mechanisms investigated include low-energy collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD), and electron-capture dissociation (ECD); all were performed on a hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer. We show that ECD, but neither CID nor IRMPD, of ADP-ribosylated Kemptide produces tandem mass spectra that are interpretable with regard to amino acid sequence assignment and site of modification. Examination of CID and IRMPD tandem mass spectra of ADP-ribosylated Kemptide revealed that fragmentation was primarily focused to the ADP-ribose region, generating several potential diagnostic ions for use in discovery of ADP-ribosylated proteins. Because of the lower relative sensitivity of ECD during data-dependent acquisition to CID, we suggest a 2-fold strategy where CID and IRMPD are first used to detect ADP-ribosylated peptides, followed by sequence assignment and location of modification by ECD analysis.


Electron Capture Dissociation Tandem Mass Spectrum Ylated Infrared Multiphoton Dissociation Kemptide 
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  1. 1.
    Corda, D.; Di Girolamo, M. Functional Aspects of Protein Mono-ADP-Ribosylation. EMBO J. 2003, 22, 1953–1958.CrossRefGoogle Scholar
  2. 2.
    Paone, G.; Wada, A.; Stevens, L. A.; Matin, A.; Hirayama, T.; Levine, R. L.; Moss, J. ADP Ribosylation of Human Neutrophil Peptide-1 Regulates Its Biological Properties. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 8231–8235.CrossRefGoogle Scholar
  3. 3.
    Coye, L. H.; Collins, C. M. Identification of SpyA, a Novel ADP-Ribosyltransferase of Streptococcus pyogenes. Mol. Microbiol. 2004, 54, 89–98.CrossRefGoogle Scholar
  4. 4.
    Huddleston, M. J.; Annan, R. S.; Bean, M. F.; Carr, S. A. Selective Detection of Phosphopeptides in Complex-Mixtures by Electrospray Liquid-Chromatography Mass-Spectrometry. J. Am. Soc. Mass Spectrom. 1993, 4, 710–717.CrossRefGoogle Scholar
  5. 5.
    Hunter, A. P.; Games, D. E. Chromatographic and Mass-Spectrometric Methods for the Identification of Phosphorylation Sites in Phosphoproteins. Rapid Commun. Mass Spectrom. 1994, 8, 559–570.CrossRefGoogle Scholar
  6. 6.
    Dookeran, N. N.; Yalcin, T.; Harrison, A. G. Fragmentation Reactions of Protonated alpha-Amino Acids. J. Mass Spectrom. 1996, 31, 500–508.CrossRefGoogle Scholar
  7. 7.
    Spahr, C. S.; Davis, M. T.; McGinley, M. D.; Robinson, J. H.; Bures, E. J.; Beierle, J.; Mort, J.; Courchesne, P. L.; Chen, K.; Wahl, R. C.; Yu, W.; Luethy, R.; Patterson, S. D. Towards Defining the Urinary Proteome Using Liquid Chromatography-Tandem Mass Spectrometry: I. Profiling an Unfractionated Tryptic Digest. Proteomics 2001, 1, 93–107.CrossRefGoogle Scholar
  8. 8.
    Margarit, S. M.; Davidson, W.; Frego, L.; Stebbins, C. E. A Steric Antagonism of Actin Polymerization by a Salmonella Virulence Protein. Structure 2006, 14, 1219–1229.CrossRefGoogle Scholar
  9. 9.
    Kharadia, S. V.; Graves, D. J. Relationship of Phosphorylation and ADP-Ribosylation Using a Synthetic Peptide as a Model Substrate. J. Biol. Chem. 1987, 262, 17379–17383.Google Scholar
  10. 10.
    Yi, E. C.; Lee, H.; Aebersold, R.; Goodlett, D. R. A Microcapillary Trap Cartridge-Microcapillary High-Performance Liquid Chromatography Electrospray Ionization Emitter Device Capable of Peptide Tandem Mass Spectrometry at the Attomole Level on an Ion Trap Mass Spectrometer with Automated Routine Operation. Rapid Commun. Mass Spectrom. 2003, 17, 2093–2098.CrossRefGoogle Scholar
  11. 11.
    Domon, B.; Costello, C. E. A Systematic Nomenclature for Carbohydrate Fragmentations in FAB- MS/MS Spectra of Glycoconjugates. Glycoconj. J. 1988, 5, 13.CrossRefGoogle Scholar
  12. 12.
    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.CrossRefGoogle Scholar
  13. 13.
    Kelleher, N. L.; Zubarev, R. A.; Bush, K.; Furie, B.; Furie, B. C.; McLafferty, F. W.; Walsh, C. T. Localization of Labile Posttranslational Modifications by Electron Capture Dissociation: The Case of gamma-Carboxyglutamic Acid. Anal. Chem. 1999, 71, 4250–4253.CrossRefGoogle Scholar
  14. 14.
    Kjeldsen, F.; Haselmann, K. F.; Budnik, B. A.; Sorensen, E. S.; Zubarev, R. A. Complete Characterization of Posttranslational Modification Sites in the Bovine Milk Protein PP3 by Tandem Mass Spectrometry with Electron Capture Dissociation as the Last Stage. Anal. Chem. 2003, 75, 2355–2361.CrossRefGoogle Scholar
  15. 15.
    Scherl, A.; Shaffer, S. A.; Taylor, G. K.; Hernandez, P.; Appel, R. D.; Binz, P. A.; Goodlett, D. R. On the Benefits of Acquiring Peptide Fragment Ions at High Measured Mass Accuracy. J. Am. Soc. Mass Spectrom. 2008, 19, 891–901.CrossRefGoogle Scholar
  16. 16.
    Crowe, M. C.; Brodbelt, J. S. Infrared Multiphoton Dissociation (IRMPD) and Collisionally Activated Dissociation of Peptides in a Quadrupole Ion Trap with Selective IRMPD of Phosphopeptides. J. Am. Soc. Mass Spectrom. 2004, 15, 1581–1592.CrossRefGoogle Scholar
  17. 17.
    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.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2009

Authors and Affiliations

  • Shawna M. Hengel
    • 1
  • Scott A. Shaffer
    • 1
  • Brook L. Nunn
    • 1
  • David R. Goodlett
    • 1
  1. 1.Department of Medicinal ChemistryUniversity of WashingtonSeattleUSA

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