Effectiveness of low energy collisional activation methods for automated peptide sequencing by tandem mass spectrometry

The relative efficiencies of low energy (< 100 eV) collisional activation techniques were determined for peptide sequencing by tandem mass spectrometry (MS/MS). Tryptic peptides were fragmented using either collision induced dissociation (CID) or surface induced dissociation (SID) to generate spectral databases. Statistical analysis of the resulting fragment ions and success rates for automated peptide sequencing by publicly available algorithms provided a measure of the value of sequencing information content of CID and SID MS/MS spectra. Typical success rates were determined for automated sequencing by SEQUEST¹⁻³, MS-Tag⁴, Mascot's⁵ MS/MS Ion Search and Sequence Query using low energy CID spectra. The ability of an algorithm to match peptide sequences to raw MS/MS data directly depended on various factors including the mass spectrometer from which the spectra were acquired, the precursor ion charge state, and the mass accuracy and resolution available in the spectra. Statistical analysis demonstrated the presence of similar fragment ions in SID and CID spectra. A lack of long contiguous ion series in SID spectra prevented automated sequencing using conventional approaches. Thus, "patchwork peptide sequencing"⁶, an unconventional method to derive specific sequence criteria for unknown peptides from SID spectra, was performed. Submitting this information to Mascot's Sequence Query allowed database searching strategies to achieve automated peptide sequencing with SID spectra. Some aspects of gas phase ion chemistry were explored for the unexpected formation of fragment ions from cleavage C-terminal to proline residues. Semi-empirical calculations suggest the most stable structure for valineprolyl-b₂ ion is a diketopiperazine, although the MS/MS/MS fragmentation pattern for VP-b₂ is indicative of an oxazalone structure. Support for a diketopiperazine structure is provided by similar fragmentation patterns for VP-b₂ and a synthetic diketopiperazine VP, and prevention of the formation of VP-b₂ by acetylation of the free N-terminus. Substitution of N-methyl alanine for proline produces a b₂ ion that fragments by loss of a portion of the valine residue, consistent with an oxazalone structure. However, theoretical calculations suggest the N-methyl alanine-containing b₂ ion is a diketopiperazine. The differences in fragmentation patterns indicate that the gas phase ion structures for the proline- and N-methyl alanine-containing b₂ ions are different, although stability calculations suggest otherwise