Repeatability and reproducibility of product ion abundances in electron capture dissociation mass spectrometry of peptides
Site-specific reproducibility and repeatability of electron capture dissociation (ECD) in Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) are of fundamental importance for product ion abundance (PIA)-based peptide and protein structure analysis. However, despite the growing interest in ECD PIA-based applications, these parameters have not yet been investigated in a consistent manner. Here, we first provide a detailed description of the experimental parameters for ECD-based tandem mass spectrometry performed on a hybrid linear ion trap (LTQ) FT-ICR MS. In the following, we describe the evaluation and comparison of ECD and infrared multiphoton dissociation (IRMPD) PIA methodologies upon variation of a number of experimental parameters, for example, cathode potential (electron energy), laser power, electron and photon irradiation periods and pre-irradiation delays, as well as precursor ion number. Ranges of experimental parameters that yielded an average PIA variation below 5% and 15% were determined for ECD and IRMPD, respectively. We report cleavage site-dependent ECD PIA variation below 20% and correlation coefficients between fragmentation patterns superior to 0.95 for experiments performed on three FT-ICR MS instruments. Overall, the encouraging results obtained for ECD PIA reproducibility and repeatability support the use of ECD PIA as a complementary source of information to m/z data in radical-induced dissociation applied for peptide and protein structure analysis.
Keywords: electron capture dissociation (ECD) ; infrared multiphoton dissociation (IRMPD) ; electrospray ionization (ESI) ; tandem MS (MS/MS) ; Fourier transform mass spectrometry (FTMS) ; ion cyclotron resonance (ICR) ; product ion abundance (PIA) ; repeatability ; reproducibility ; variability ; Collision-Induced Dissociation ; Charged Protein Cations ; Cyclotron Resonance ; Internal Energy ; Magnetron Motion ; Spectra ; Prediction ; Fragmentation ; Ionization ; Mechanism
Record created on 2011-10-24, modified on 2016-08-09