Unimolecular Fragmentation of Deprotonated Diproline [Pro2-H]- Studied by Chemical Dynamics Simulations and IRMPD Spectroscopy.

Dissociation chemistry of the diproline anion [Pro2-H]- is studied using chemical dynamics simulations coupled with quantum-chemical calculations and RRKM analysis. Pro2- is chosen due to its reduced size and the small number of sites where deprotonation can take place. The mechanisms leading to the two dominant collision-induced dissociation (CID) product ions are elucidated. Trajectories from a variety of isomers of [Pro2-H]- were followed in order to sample a larger range of possible reactivity. While different mechanisms yielding y1- product ions are proposed, there is only one mechanism yielding the b2- ion. This mechanism leads to formation of a b2- fragment with a diketopiperazine structure. The sole formation of a diketopiperazine b2 sequence ion is experimentally confirmed by infrared ion spectroscopy of the fragment anion. Furthermore, collisional and internal energy activation simulations are used in parallel to identify the different dynamical aspects of the observed reactivity.

Deamidation reactions of protonated asparagine and glutamine investigated by ion spectroscopy.

RATIONALE: Deamidation of Asn and Gln residues is a primary route for spontaneous post-translational protein modification. Several 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(+) -NH3](+) is found to possess a 3-aminosuccinic anhydride structure protonated on the amino group. The dissociation reaction involving loss of H2O and CO forms a linear immonium ion. For [GlnH(+)-NH3](+), the N-terminal nitrogen acts as the nucleophile leading to an oxo-proline product ion structure. For [GlnH(+)-NH3](+), a sequential loss of [CO + H2O] 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 NH3 from AsnH(+) and GlnH(+). CONCLUSIONS: The structure determined for AsnH(+) is in agreement with the suggested structure derived from measured and computed activation energies. IR ion spectra for the NH3 -loss product from GlnH(+) establish that a different reaction mechanism occurs for this species as compared to AsnH(+). For both amino acids, loss of NH3 occurs from the side chain.

Structural identification of electron transfer dissociation products in mass spectrometry using infrared ion spectroscopy.

Tandem mass spectrometry occupies a principle place among modern analytical methods and drives many developments in the 'omics' sciences. Electron attachment induced dissociation methods, as alternatives for collision-induced dissociation have profoundly influenced the field of proteomics, enabling among others the top-down sequencing of entire proteins and the analysis of post-translational modifications. The technique, however, produces more complex mass spectra and its radical-driven reaction mechanisms remain incompletely understood. Here we demonstrate the facile structural characterization of electron transfer dissociation generated peptide fragments by infrared ion spectroscopy using the tunable free-electron laser FELIX, aiding the elucidation of the underlying dissociation mechanisms. We apply this method to verify and revise previously proposed product ion structures for an often studied model tryptic peptide, [AlaAlaHisAlaArg+2H](2+). Comparing experiment with theory reveals that structures that would be assigned using only theoretical thermodynamic considerations often do not correspond to the experimentally sampled species.

Deamidation Reactions of Asparagine- and Glutamine-Containing Dipeptides Investigated by Ion Spectroscopy.

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 Abstract ᅟ.

Spectroscopic identification of cyclic imide b2-ions from peptides containing Gln and Asn residues.

In mass-spectrometry based peptide sequencing, formation of b- and y-type fragments by cleavage of the amide C-N bond constitutes the main dissociation pathway of protonated peptides under low-energy collision induced dissociation (CID). The structure of the b2 fragment ion from peptides containing glutamine (Gln) and asparagine (Asn) residues is investigated here by infrared ion spectroscopy using the free electron laser FELIX. The spectra are compared with theoretical spectra calculated using density functional theory for different possible isomeric structures as well as to experimental spectra of synthesized model systems. The spectra unambiguously show that the b2-ions do not possess the common oxazolone structure, nor do they possess the alternative diketopiperazine structure. Instead, cyclic imide structures are formed through nucleophilic attack by the amide nitrogen atom of the Gln and Asn side chains. The alternative pathway involving nucleophilic attack from the side-chain amide oxygen atom leading to cyclic isoimide structures, which had been suggested by several authors, can clearly be excluded based on the present IR spectra. This mechanism is perhaps surprising as the amide oxygen atom is considered to be the better nucleophile; however, computations show that the products formed via attack by the amide nitrogen are considerably lower in energy. Hence, b2-ions with Asn or Gln in the second position form structures with a five-membered succinimide or a six-membered glutarimide ring, respectively. b2-Ions formed from peptides with Asn in the first position are spectroscopically shown to possess the classical oxazolone structure.

Structures of a(n)* ions derived from protonated pentaglycine and pentaalanine: results from IRMPD spectroscopy and DFT calculations.

Infrared multiple-photon dissociation (IRMPD) spectroscopy and DFT calculations have been used to probe the most stable structures of a3(*) and a4(*) ions derived from both protonated pentaglycine (denoted G5) and pentaalanine (A5). The a3(*) and a4(*) ions derived from protonated A5 feature a CHR=N-CHR'- group at the N-terminus and an oxazolone ring at the C-terminus, as proposed previously [J. Am. Soc. Mass Spectrom. 19, 1788-1798 (2008)]. The isomeric a4(*) ion derived from A5 with a 3,5-dihydro-4H-imidazol-4-one ring structure was calculated to have a slightly better energy than the oxazolone, but the barrier to its formation is higher and there was no evidence of this ion in the IRMPD spectrum. By contrast, the a4(*) and [a4 - H2O](+) (denoted a4(0)) ions from G5 gave strikingly similar IRMPD spectra and both have the 3,5-dihydro-4H-imidazol-4-one ring structure similar to that recently reported for the [GGGG + H - H2O](+) ion [Int. J. Mass Spectrom. 316-318, 268-272 (2012)]. In the absence of a solvent molecule, the pathway to the oxazolone is calculated to be lower than those to thermodynamically more stable products, the a4(0) and the a4(*) with the 3,5-dihydro-4H-imidazol-4-one ring structure. Incorporation of one water molecule is sufficient to reduce the barrier to formation of the a4(0) of G5 to below that for formation of the oxazolone. On the equivalent potential energy surface for protonated A5 the barrier to formation of the a4(0) ion is 12.3 kcal mol(-1) higher than that for oxazolone formation and the a4(0) ion is not observed experimentally.

Structure and reactivity of the distonic and aromatic radical cations of tryptophan.

In this work, we regiospecifically generate and compare the gas-phase properties of two isomeric forms of tryptophan radical cations-a distonic indolyl N-radical (H3N(+) - TrpN(•)) and a canonical aromatic π (Trp(•+)) radical cation. The distonic radical cation was generated by nitrosylating the indole nitrogen of tryptophan in solution followed by collision-induced dissociation (CID) of the resulting protonated N-nitroso tryptophan. The π-radical cation was produced via CID of the ternary [Cu(II)(terpy)(Trp)](•2+) complex. CID spectra of the two isomeric species were found to be very different, suggesting no interconversion between the isomers. In gas-phase ion-molecule reactions, the distonic radical cation was unreactive towards n-propylsulfide, whereas the π radical cation reacted by hydrogen atom abstraction. DFT calculations revealed that the distonic indolyl radical cation is about 82 kJ/mol higher in energy than the π radical cation of tryptophan. The low reactivity of the distonic nitrogen radical cation was explained by spin delocalization of the radical over the aromatic ring and the remote, localized charge (at the amino nitrogen). The lack of interconversion between the isomers under both trapping and CID conditions was explained by the high rearrangement barrier of ca.137 kJ/mol. Finally, the two isomers were characterized by infrared multiple-photon dissociation (IRMPD) spectroscopy in the ~1000-1800 cm(-1) region. It was found that some of the main experimental IR features overlap between the two species, making their distinction by IRMPD spectroscopy in this region problematic. In addition, DFT theoretical calculations showed that the IR spectra are strongly conformation-dependent.

Spectroscopic evidence for an oxazolone structure in anionic b-type peptide fragments.

Infrared spectra of anionic b-type fragments generated by collision induced dissociation (CID) from deprotonated peptides are reported. Spectra of the b(2) fragments of deprotonated AlaAlaAla and AlaTyrAla have been recorded over the 800-1800 cm(-1) spectral range by multiple-photon dissociation (MPD) spectroscopy using an FTICR mass spectrometer in combination with the free electron laser FELIX. Structural characterization of the b-type fragments is accomplished by comparison with density functional theory calculated spectra at the B3LYP/6-31++G(d,p) level for different isomeric structures. Although diketopiperazine structures represent the energetically lowest isomers, the IR spectra suggest an oxazolone structure for the b(2) fragments of both peptides. Deprotonation is shown to occur on the oxazolone α-carbon, which leads to a conjugated structure in which the negative charge is practically delocalized over the entire oxazolone ring, providing enhanced gas-phase stability.

Non-Equilibrium Isomer Distribution of the Gas-Phase Photoactive Yellow Protein Chromophore.

The conjugate base of para-coumaric acid, which can be conveniently generated in the gas phase by electrospray ionization (ESI), is a commonly used model system for the chromophore of the photoactive yellow protein. Here we report its gas-phase IR spectrum, which shows that the anion easily adopts a carboxylate structure lying 60 kJ/mol higher in energy than the global minimum phenoxide structure. Generation of the biologically more relevant phenoxide isomer by ESI can be achieved using dry acetonitrile as solvent.