Data-dependent electron capture dissociation FT-ICR mass spectrometry for proteomic analyses.
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-ECD-MS/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 high-throughput proteomic strategies.
Cooper
Helen J
HJ
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. h.j.cooper@bham.ac.uk
Akbarzadeh
Shiva
S
Heath
John K
JK
Zeller
Martin
M
D000465:Algorithms; D000595:Amino Acid Sequence; D019902:Chondrocytes / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D019992:Databases as Topic; D030541:Databases, Genetic; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D010455:Peptides; D020543:Proteome; D014357:Trypsin