No abstract

Deamidation is a major fragmentation channel upon activation by collision induced dissociation (CID) for protonated peptides containing glutamine (Gln) and asparagine (Asn) residues. Here, we investigate these NH3-loss reactions for four Asn- and Gln-containing protonated peptides in terms of the resulting product ion structures using infrared ion spectroscopy with the free electron laser FELIX. The influence of the side chain length (Asn versus Gln) and of the amino acid sequence on the deamidation reaction has been examined. Molecular structures for the product ions are determined by comparison of experimental IR spectra with spectra predicted by density functional theory (DFT). The reaction mechanisms identified for the four dipeptides AlaAsn, AsnAla, AlaGln, and GlnAla are not the same. For all four dipeptides, primary deamidation takes place from the amide side chain (and not from the N-terminus) and, in most cases, resembles the mechanisms previously identified for the protonated amino acids asparagine and glutamine. Secondary fragmentation reactions of the deamidation products have also been characterized and provide further insight in – and confirmation of – the identified mechanisms. Overall, this study provides a comprehensive molecular structure map of the deamidation chemistry of this series of dipeptides. Graphical Abstractᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-016-1462-5) contains supplementary material, which is available to authorized users.

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.

RATIONALE:Deamidation of Asn and Gln residues is a primary route for spontaneous posttranslational protein modification. Various structures have been proposed for the deamidation products of the protonated amino acids. Here we verify these structures by ion spectroscopy, as well as the structures of parallel and sequential fragmentation products. METHODS:Infrared ion spectroscopy using the free electron laser FELIX has been applied to the reaction products from deamidation of protonated glutamine and asparagine in a tandem mass spectrometer. IR spectra were recorded over the 800-1900 cm -1 spectral range by infrared multiple-photon dissociation (IRMPD) spectroscopy. Molecular structures of the fragment ions are derived from comparison of the experimental spectra with spectra predicted for different candidate structures by density functional theory (DFT) calculations. RESULTS:[AsnH + -NH 3 ] + is found to possess a 3-amino succinic anhydride structure protonated on the amino group. The dissociation reaction involving loss of H 2 O and CO forms a linear immonium ion. For [GlnH + -NH 3 ] + , the N-terminal nitrogen acts as the nucleophile leading to an oxo-proline product ion structure. For [GlnH + -NH 3 ] + a sequential loss of [CO + H 2 O] is found, leading to a pyrolidone-like structure. We also confirm by IR spectroscopy that dehydration of protonated aspartic acid (AspH + ) and glutamic acid (GluH + ) leads to identical structures as to those found for the loss of NH 3 from AsnH + and GlnH + .2 CONCLUSIONS: The structure determined for AsnH + is in agreement with the suggested structure derived from measured and computed activation energies. Infrared ion spectra for the NH 3 -loss product from GlnH + establish that a different reaction mechanism occurs for this species as compared to AsnH + . For both amino acids, loss of NH 3 occurs from the side-chain.

The molecular structures of six open-shell z3-ions resulting from electron transfer dissociation mass spectrometry (ETD MS) were investigated using infrared ion spectroscopy in combination with density functional theory and molecular mechanics/molecular dynamics calculations.