Using dissociation energies to predict observability of b‐ and y‐peaks in mass spectra of short peptides

Abstract

RATIONALE Peptide identification reliability can be improved by excluding from analysis those m/z peaks of candidate peptides, which can not be observed in practice due to various physical, chemical or thermodynamic considerations. We propose using dissociation energies (as opposed to proton affinities) as a predictor of observability of different m/z peaks in spectra of short peptides. METHODS Experiment Mass spectra of tetrapeptides AAAA, AAFA, AAVA, AFAA, AVAA, AFFA, AVVA have been measured in the CID activation mode on a grid of activation times 0.05 to 100 ms and normalized collision energy 10 to 35 %. Computations Lowest energy geometries and vibrational spectra were calculated for the precursor ions and their charged and neutral fragments using DFT at the TPSS/6-31G(d,p) level. Dissociation energies were calculated for all fragmentation channels leading to b− or y− fragments. RESULTS It is demonstrated that m/z peaks observed in the mass spectra correspond to the fragmentation channels with the lowest dissociation energies. Using 50 kcal/mol as the cut off value of dissociation energy, it was predicted that 28 out of 42 possible peaks in the b− and y− series of the seven tetrapeptides cab be observed in mass spectra. In the experiments, 26 b− or y− peaks were observed, all of which are among the 28 predicted ones. CONCLUSIONS The use of dissociation energies generalizes the use of proton affinities for semi-quantitative predictions of relative intensities of different m/z peaks of short peptides. Further advances in this direction will pave the way for reliable quantitative predictions and, hence, for a significant improvement in robustness and accuracy of peptide and protein identification tools.

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

“…The experimental set-up was identical to the one used in our experiments with tetrapeptides. [22] Namely, experiments were performed on a Finnigan LTQ XL mass spectrometer in the collision-induced dissociation (CID) activation mode. The product ion spectra were acquired on a grid of activation times t a varying from 0.05 to 100 ms and normalized collision energy (NCE) varying from 10 to 35%.…”

“…Finally, we note that knowledge of dissociation pathways is very important. As is discussed in the first part of our study, [22] the geometries of the charged and neutral fragments to be used for calculating the dissociation energies are not always the lowest energy geometries. For example, neutral b-fragments can be oxazolone or diketopiperazine derivatives.…”

“…As in our previous study, [22] numerical calculations were carried out at the TPSS/6-31G(d,p) level of the density functional theory (DFT) with the use of the Gaussian 09 software package [23] on the Biowulf computer cluster at the National Institutes of Health.…”

“…The ratio of intensities I 1 and I 2 for any pair of fragmentation channels 1 and 2 with dissociation energies E 1 and E 2 is assumed to be predicted from calculated energy differences (see the first part of our study [22] for a discussion of the assumptions and for references):…”

“…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.…”