Patchwork peptide sequencing: extraction of sequence information from accurate mass data of peptide tandem mass spectra recorded at high resolution.

The accurate mass values of all immonium, y(1), y(2), a(2), and b(2) ions of tryptic peptides composed of the 20 standard amino acids were calculated. The differences between adjacent masses in this data set are greater than 10 mDa for more than 80% of the values. Using this mass list, the majority of low mass ions in quadrupole-time of flight tandem mass spectra of peptides from tryptic digests and from an elastase digest could be assigned. Besides the a(2)/b(2) ions, which carry residues 1-2 from the N-terminus, a variety of internal dipeptide b ions were regularly observed. In case internal proline was present, corresponding dipeptide b ions carrying proline at the N-terminal position occurred. By assigning the dipeptide b ions on the basis of their accurate mass, bidirectional or unidirectional sequence information was obtained, which is localized to the peptide N-terminus (a(2)/b(2) ions) or not localized (internal b ions). Identification of the y(1) and y(2) ions by their accurate mass provides unidirectional sequence information localized to the peptide C-terminus. It is shown that this patchwork-type sequence information extractable from accurate mass data of low-mass ions is highly efficient for protein identification.