Complete sequence confirmation of large peptides by high energy collisional activation of multiply protonated ions

High energy collisional activation on a tandem four-sector mass spectrometer produces fragmentation throughout the peptide backbone of multiply protonated peptides in the 4-5-ku molecular weight range, which enables complete sequence confirmation from these peptides, including leucine-isoleucine determinations. The resolutions provided by this type of mass spectrometer permit the charge state of the fragment ions to be determined by identifying their isotopic spacing. This eliminates the complexity in spectral interpretation caused by the presence of fragment ions in various charge states. The reliable mass assignments from a sector mass spectrometer permit identification of amino acids that differ by as little as a single unit in these large peptides. The combination of excellent mass accuracy with the ability to assign charge states is expected to greatly facilitate the interpretation of tandem mass spectra from unknown peptides, and permit their sequences to be deduced. These results lead us to propose tandem mass spectrometric analysis of multiply charged precursor ions on a tandem four-sector instrument as a direct strategy for determination of the complete amino acid sequence of peptides up to 5 ku.