Data-Dependent Electron Capture Dissociation FT-ICR Mass Spectrometry for Proteomic Analyses Helen J. Cooper,*,† Shiva Akbarzadeh,† John K. Heath,† and Martin Zeller‡ School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom, and Thermo Electron Corporation, Hanna-Kunath-Str. 11, 28199 Bremen, Germany Received April 6, 2005
Electron capture dissociation (ECD) offers many benefits for the analysis of peptides and proteins, and consequently shows great potential for the field of proteomics. Recent developments have reduced the time scale required for ECD to milliseconds resulting in the technique’s compatibility with on-line separation techniques, e.g., HPLC. Here, we demonstrate incorporation of ECD into a high-throughput data-dependent LC-MS/MS approach for the analysis of proteomic samples. The approach is applied to analysis of the protein Fc-ROR2 isolated from chondrocytes and is the first example of LC-ECDMS/MS of such a sample. Protein sequence coverage was 29%. Within that coverage, fifteen peptides were isolated and subjected to ECD. In most cases, the sequence tag generated by ECD was over 70% (in terms of the number of peptide backbone cleavages). The ECD data were searched against the nonredundant human NCBI database using the SEQUEST algorithm. Protein ROR2 was assigned, as was IgG (Fc domain). The results demonstrate the suitability of ECD as an integral technique in highthroughput proteomic strategies. Keywords: ECD • FTICR • FT-MS • proteomics • LC-MS/MS
Introduction Electron capture dissociation (ECD)1 coupled to Fourier transform ion cyclotron resonance mass spectrometry (FTICR)2 is a recently developed tandem mass spectrometry (MS/ MS) technique that offers many advantages for the analysis of peptides and proteins, and consequently shows great potential for the field of proteomics. Traditional MS/MS techniques, the so-called “slow-heating” techniques, such as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD),3 result in slow activation of the precursor ions and fragment ions form via the lowest energy pathway(s). In peptides and proteins, cleavage of the peptide N-CO bond results in b and y fragment ions.4 In ECD, trapped ions are irradiated with low energy (
