Deamidation reactions of protonated asparagine and glutamine investigated by ion spectroscopy

Abstract

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.

Dehydration reactions of protonated dipeptides containing asparagine or glutamine investigated by infrared ion spectroscopy

“…These structures were then optimized using the DFT protocol and their scaled harmonic spectra were compared with the experimental spectra. The computational procedure is described in more detail elsewhere [25,41,44].…”

“…Structures of the product ions were determined using a combination of mass spectrometry, infrared multiple photon dissociation (IRMPD) spectroscopy and theoretical calculations. IRMPD spectroscopy has previously been used to determine the structures of several protonated peptide precursor ions and fragments [17][18][19][20][21][22], and has proven to be a powerful method to distinguish isomers, (prototropic) tautomers and conformers [18,21,[23][24][25][26][27][28][29][30][31][32][33].…”

Synthesis and Hydrolysis of Uranyl, Neptunyl, and Plutonyl Gas-Phase Complexes Exhibiting Discrete Actinide–Carbon Bonds

“…Hydrolysis in the ion trap produced hydroxides for spectroscopic studies. The QIT/MS has been modified 37 such that the high-intensity tunable IR beam from FELIX can be directed into the ion packet, resulting in multiphoton dissociation that is appreciable only when the IR frequency is in resonance with an adequately high absorption vibrational mode of the particular mass-selected complex being studied. The FEL produces ∼5 μs long IR pulses with an energy of typically 40 mJ, which are in the form of a sequence of ∼1 ps long − decarboxylation energies as well as possible competitive reaction energies (i.e., ligand elimination) for R = CH 3 (methyl), CH 3 CC (1-propynyl), C 6 H 5 (phenyl), C 6 F 5 (pentafluorophenyl).…”

w‐Type ions formed by electron transfer dissociation of Cys‐containing peptides investigated by infrared ion spectroscopy

“…Single‐point electronic energies were also obtained at the MP2(full)/6‐31 + G(d,p)//B3LYP/6‐31++G(d,p) level for comparison. The computational procedure is described in more detail elsewhere …”