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

Abstract

MS-based proteomics usually involves the fragmentation of tryptic peptides (tandem MS or MS(2)) 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 MS(3). Here, we report that efficient ion capture in a linear ion trap leads to MS(3) acquisition times and spectra quality similar to those for MS(2) 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 MS(3) spectrum greatly constrains the C-terminal amino acids of the peptide sequence. MS(3) 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 MS(3) spectra per peptide can be obtained even when sequencing very complex mixtures. We calculate the probability that an experimental MS(3) spectrum originates from fragmentation of a given N- or C-terminal ion of a peptide under consideration. This MS(3) identification score can be combined with the MS(2) scores of the precursor peptide from existing search engines. When MS(3) is performed on the linear ion trap-Fourier transform mass spectrometer combination, accurate peptide masses further increase confidence in peptide identification.