Tandem mass spectrometry investigation of ADP-ribosylated kemptide
- 213 Downloads
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.
- 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
- 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.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
- 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