Improving Protein Identification Using Complementary Fragmentation Techniques in Fourier Transform Mass Spectrometry

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 (Ϸ1 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. Molecular & Cellular Proteomics 4:835-845, 2005.

Bifurcating fragmentation behavior of gas-phase tryptic peptide dications in collisional activation

“…The peptides were identified by the Mascot search engine (Matrix Sciences, London, UK). The details of the experimental procedure and data processing are described in Nielsen and colleagues [9] and Savitski and colleagues [10].…”

Effects of Peptide Backbone Amide-to-Ester Bond Substitution on the Cleavage Frequency in Electron Capture Dissociation and Collision-Activated Dissociation

“…This complementarity allows for more reliable protein identification when both CAD and ECD/ETD techniques are applied to the same polypeptide [11]. Analytical benefits of ECD/ETD in proteome research have been demonstrated in a number of studies [12,13].…”

Towards liquid chromatography time-scale peptide sequencing and characterization of post-translational modifications in the negative-ion mode using electron detachment dissociation tandem mass spectrometry

“…EDD produces predominantly a-and x-ions, which are complementary ions to both ECD/ETD (c-and z-ions) and CAD (b-and y-ions) produced in the positive-ion mode. Generation of complementary ion pairs increases the confidence of peptide identification [38]. In this study we demonstrated that simple optimization of instrumental parameters is sufficient to increase the fragmentation efficiency of EDD many-fold, making EDD compatible with LC-MS/MS and peptide sequencing at the chromatographic time-scale.…”