Deamidation of Protonated Asparagine–Valine Investigated by a Combined Spectroscopic, Guided Ion Beam, and Theoretical Study

Abstract

Peptide deamidation of asparaginyl residues is a spontaneous post-translational modification that is believed to play a role in aging and several diseases. It is also a well-known small-molecule loss channel in the MS/MS spectra of protonated peptides. Here we investigate the deamidation reaction, as well as other decomposition pathways, of the protonated dipeptide asparagine–valine ([AsnVal + H]+) upon low-energy activation in a mass spectrometer. Using a combination of infrared ion spectroscopy, guided ion beam tandem mass spectrometry, and theoretical calculations, we have been able to identify product ion structures and determine the energetics and mechanisms for decomposition. Deamidation proceeds via ammonia loss from the asparagine side chain, initiated by a nucleophilic attack of the peptide bond oxygen on the γ-carbon of the Asn side chain. This leads to the formation of a furanone ring containing product ion characterized by a threshold energy of 129 ± 5 kJ/mol (15 kJ/mol higher in energy than dehydration of [AsnVal + H]+, the lowest energy dissociation channel available to the system). Competing formation of a succinimide ring containing product, as has been observed for protonated asparagine–glycine ([AsnGly + H]+) and asparagine–alanine ([AsnAla + H]+), was not observed here. Quantum-chemical modeling of the reaction pathways confirms these subtle differences in dissociation behavior. Measured reaction thresholds are in agreement with predicted theoretical reaction energies computed at several levels of theory.

Influence of a Hydroxyl Group on the Deamidation and Dehydration Reactions of Protonated Asparagine-Serine Investigated by Combined Spectroscopic, Guided Ion Beam, and Theoretical Approaches

“…Therefore, in the current work, we establish the likely mechanisms for possible deamidation processes of protonated AsnSer ([AsnSer+H] + ), identify the deamidation product(s) formed, and evaluate the reaction threshold for deamidation via infrared (IR) action spectroscopy and threshold collision-induced dissociation (TCID) experiments, coupled with theory. This work complements analogous studies conducted previously on [AsnXxx+H] + for Xxx = Gly, 16 Ala, 17,18 Val, 19 and Thr. 20 Succinimide intermediate formation as a result of Asn deamidation is well-documented in the literature; 1,2,21 however, given the intrinsic differences between a dipeptide and a protein as well as between gas-phase and condensedphase reactivity, we have also explored alternative pathways that may exist for ammonia loss.…”

“…16 The deamidation of [AsnAla+H] + proceeds along bifurcating pathways resulting in the observation of both Fur and Suc structures, 17,18 whereas the deamidation of [AsnVal+H] + results exclusively in the formation of a Fur structure. 19 In contrast, the deamidation of [AsnThr+H] + leads predominantly to the formation of a Suc product ion, 20 with a minor contribution of Fur. Thus, the current results, compared with our previous analyses of [AsnGly+H] + , [AsnAla+H] + , [AsnVal +H] + , and [AsnThr+H] + , should allow for the determination of C-terminal residue side-chain effects with respect to both size and side-chain functionality.…”

Relative Energetics of the Gas Phase Protomers of p-Aminobenzoic Acid and the Effect of Protonation Site on Fragmentation

“…Indeed, tightening the vibrational frequencies below 900 cm –1 (those tagged by Gaussian 16 as potentially inaccurate) of the tight TSs by ∼25% is sufficient to bring the TCID thresholds into agreement with the theoretical values for H 2 O and CO 2 loss from (O) p-ABAH + and for H 2 O + CO 2 loss from (N)-p-ABAH + . This extent of scaling for TS frequencies is precedented in several studies conducted on the dissociation behavior of small protonated di- and tripeptides, where scaling factors of 22–32% were needed in order to quantitatively predict the relative magnitudes and energetics for competing dissociation pathways. ,− For BAH + , the low-frequencies for the TSs leading to H 2 O and CO 2 loss require tightening by ∼50% in order to yield quantitative agreement.…”

Water Loss from Protonated XxxSer and XxxThr Dipeptides Gives Oxazoline—Not Oxazolone—Product Ions

“…Various (spectroscopic) studies on the dehydration of protonated dipeptides not containing a Ser or Thr residue showed that water is expelled from the C-terminal carboxylic acid group. ,,,, This reaction is suggested to proceed via a nucleophilic attack on the C-terminal carboxylic acid carbon generating a b 2 -type sequence ion, which can have either an oxazolone or a diketopiperazine structure. In contrast, for the Thr and Ser terminal peptides studied here, an alternative mechanism is at work.…”