Abstract
Identification of proteins by MS/MS is performed by matching experimental mass spectra against calculated spectra of all possible peptides in a protein data base. The search engine assigns each spectrum a score indicating how well the experimental data complies with the expected one; a higher score means increased confidence in the identification. One problem is the false-positive identifications, which arise from incomplete data as well as from the presence of misleading ions in experimental mass spectra due to gas-phase reactions, stray ions, contaminants, and electronic noise. We employed a novel technique of reduction of false positives that is based on a combined use of orthogonal fragmentation techniques electron capture dissociation (ECD) and collisionally activated dissociation (CAD). Since ECD and CAD exhibit many complementary properties, their combined use greatly increased the analysis specificity, which was further strengthened by the high mass accuracy (approximately 1 ppm) afforded by Fourier transform mass spectrometry. The utility of this approach is demonstrated on a whole cell lysate from Escherichia coli. Analysis was made using the data-dependent acquisition mode. Extraction of complementary sequence information was performed prior to data base search using in-house written software. Only masses involved in complementary pairs in the MS/MS spectrum from the same or orthogonal fragmentation techniques were submitted to the data base search. ECD/CAD identified twice as many proteins at a fixed statistically significant confidence level with on average a 64% higher Mascot score. The confidence in protein identification was hereby increased by more than 1 order of magnitude. The combined ECD/CAD searches were on average 20% faster than CAD-only searches. A specially developed test with scrambled MS/MS data revealed that the amount of false-positive identifications was dramatically reduced by the combined use of CAD and ECD.
Highlights
Identification of proteins by MS/MS is performed by matching experimental mass spectra against calculated spectra of all possible peptides in a protein data base
Masses involved in complementary pairs in the MS/MS spectrum from the same or orthogonal fragmentation techniques were submitted to the data base search
The utility of complementary fragmentation techniques in conjunction with data-dependent LC-MS/MS acquisition on a new hybrid Fourier transform ion cyclotron resonance mass spectrometer for identification of a large number of proteins has been demonstrated for the first time
Summary
Identification of proteins by MS/MS is performed by matching experimental mass spectra against calculated spectra of all possible peptides in a protein data base. Gaseous peptide cations are collided with inert gas molecules in what is termed collisionally activated dissociation (CAD), which predominantly gives rise to informative N- and C-terminal peptide fragments (so called b and y ions) as the amide backbone bonds dissociate [6, 7] This approach combined with separation of complex peptide mixtures by LC allows for identification of hundreds of proteins in one run (8 –12), but processing this enormous amount of data is not straightforward. Improving Protein Identification Using Complementary Pairs ments and false identifications can sometimes be achieved by investigating spectra manually and verifying the peptide fragment assignment Such a time-consuming approach is not possible when the data of interest contains thousands of MS/MS spectra. The current work was undertaken to test these assumptions and to evaluate the benefits and the drawbacks of the complementary pair approach
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