Tandem mass spectrometry investigation of ADP-ribosylated kemptide.
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.
Hengel
Shawna M
SM
Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195-7610, USA.
Shaffer
Scott A
SA
Nunn
Brook L
BL
Goodlett
David R
DR
D036002:ADP Ribose Transferases / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
D009842:Oligopeptides; D010446:Peptide Fragments; C037642:kemptide; D036002:ADP Ribose Transferases