Using dissociation energies to predict observability of b- and y-peaks in mass spectra of short peptides. II. Results for hexapeptides with non-polar side chains.

One of the potential applications of molecular-level studies of short peptide fragmentation mechanisms would be in silico predictions of relative intensities of various m/z peaks in a product ion mass spectrum of a given peptide. Ion intensity relationships, if they can be correctly predicted, would definitely improve selectivity and reliability of peptide identification tools. At the present level of computing power, a comprehensive computational analysis of all possible fragmentation pathways, even for diglycine, is not an easy task.[1] Moreover, accurate predictions of ion intensity relationships would also require detailed knowledge of experimental conditions (which is not always available) and also a satisfactory theoretical description of other physical processes (collisional heating, electronic excitations, thermalization, etc.).[2–7] Although accurate prediction of relative ion intensities remains a task for the future, we believe that one already can take advantage of the achieved understanding[8–21] of peptide fragmentation mechanisms. A practical way to do so would be to introduce a filtering of the theoretical peaks of candidate peptides before scoring their similarity with the given experimental spectrum. In most of the peptide identification tools, all theoretically possible b- and y-peaks of a candidate peptide are scored (often with equal weights) against the peaks present in the experimental mass spectrum. The similarity scores can be made much more accurate if the scoring function does not take into account theoretical peaks which cannot be observed in real-life product ion spectra due to various physical, chemical, or thermodynamic reasons. In order to implement such a filtering one needs to choose a physical quantity which would be experiment-independent and relatively easily computable, but which still could serve as a reliable predictor of observability of b- and y-peaks. In our previous paper[22] we suggested that the role of this observability predictor can be played by dissociation energy. We demonstrated, using seven tetrapeptides as examples, that the m/z peaks observed in experimental mass spectra correspond to the fragmentation channels with the lowest dissociation energies, and one can choose a certain cut-off energy separating observable peaks from unobservable ones. In particular, choosing 50 kcal/mol as the cut-off for dissociation energy, allowed us to correctly predict observability of 40 out of 42 theoretically possible b- and y-peaks in product ion spectra of those seven tetrapeptides. In this follow-up work we present results on employing dissociation energies for predicting observability of b- and y-peaks in product ion spectra of seven hexapeptides, AAAAAA, AAAFAA, AAAVAA, AAFAAA, AAVAAA, AAFFAA and AAVVAA. We calculated dissociation energies for 63 theoretically possible fragmentation channels which result in either b- or y-fragments. We also acquired product ion spectra of these seven hexapeptides in a wide range of collision energies and activation times. Using the same cut-off value for the dissociation energies as we did for tetrapeptides (50 kcal/mol), we predicted that in total 44 b- and y-peaks can be observed in the experimental spectra and 19 peaks should not be observed. In experiments, we observed 39 peaks, all of which were predicted as observable. More importantly for the filtering purposes, none of the 19 peaks classified as unobservable had in experimental spectra intensity greater than 1%. Therefore, our new data support the hypothesis that dissociation energies can serve as a reliable predictor of observability of b- and y-peaks in product ion m/z spectra of short peptides with non-polar side chains.