Reduction in database search space by utilization of amino acid composition information from electron transfer dissociation and higher-energy collisional dissociation mass spectra.
With high-mass accuracy and consecutively obtained electron transfer dissociation (ETD) and higher-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS), reliable (≥97%) and sensitive fragment ions have been extracted for identification of specific amino acid residues in peptide sequences. The analytical benefit of these specific amino acid composition (AAC) ions is to restrict the database search space and provide identification of peptides with higher confidence and reduced false negative rates. The 6706 uniquely identified peptide sequences determined with a conservative Mascot score of >30 were used to characterize the AAC ions. The loss of amino acid side chains (small neutral losses, SNLs) from the charge reduced peptide radical cations was studied using ETD. Complementary AAC information from HCD spectra was provided by immonium ions. From the ETD/HCD mass spectra, 5162 and 6720 reliable SNLs and immonium ions were successfully extracted, respectively. Automated application of the AAC information during database searching resulted in an average 3.5-fold higher confidence level of peptide identification. In addition, 4% and 28% more peptides were identified above the significance level in a standard and extended search space, respectively.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Thomas A
TA
Hansen
Fedor
F
Kryuchkov
Frank
F
Kjeldsen
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D016208:Databases, Factual; D004579:Electron Transport; D006367:HeLa Cells; D006801:Humans; D010455:Peptides / Q000032:analysis*; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
D010455:Peptides; D014357:Trypsin