Improved peptide identification in proteomics by two consecutive stages of mass spectrometric fragmentation

MS-based proteomics usually involves the fragmentation of tryptic peptides (tandem MS or MS2) and their identification by searching protein sequence databases. In ion trap instruments fragments can be further fragmented and analyzed, a process termed MS/MS/MS or MS3. Here, we report that efficient ion capture in a linear ion trap leads to MS3 acquisition times and spectra quality similar to those for MS2 experiments with conventional 3D ion traps. Fragmentation of N- or C-terminal ions resulted in informative and low-background spectra, even at subfemtomol levels of peptide. Typically C-terminal ions are chosen for further fragmentation, and the MS3 spectrum greatly constrains the C-terminal amino acids of the peptide sequence. MS3 spectra allow resolution of ambiguities in identification, a crucial problem in proteomics. Because of the sensitivity and rapid scan rates of the linear ion trap, several MS3 spectra per peptide can be obtained even when sequencing very complex mixtures. We calculate the probability that an experimental MS3 spectrum originates from fragmentation of a given N- or C-terminal ion of a peptide under consideration. This MS3 identification score can be combined with the MS2 scores of the precursor peptide from existing search engines. When MS3 is performed on the linear ion trap-Fourier transform mass spectrometer combination, accurate peptide masses further increase confidence in peptide identification.