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.

Keywords

Affiliated Institutions

• University of Southern Denmark DK

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