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International audience; 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.

Dissociation chemistry of the diproline anion [Pro

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.

Infrared multiple-photon dissociation (IRMPD) spectroscopy and DFT calculations have been used to probe the most stable structures of a 3 * and a 4 * ions derived from both protonated pentaglycine (denoted G5) and pentaalanine (A5). The a 3 * and a 4 * 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 a 4 * 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 a 4 * and [a 4 – H2O]+ (denoted a 4 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 a 4 0 and the a 4 * 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 a 4 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 a 4 0 ion is 12.3 kcal mol–1 higher than that for oxazolone formation and the a 4 0 ion is not observed experimentally.

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 b 2 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.

Collision-induced dissociation has been used to study the fragmentations of two protonated diketopiperazines, protonated cyclic-glycylglycine and cyclic-alanylalanine. Protonated cyclo-AA lost CO and (CO + NH3) at low collision energies, channels attributed to dissociation of the O-protonated tautomer. Higher collision energies were required to dissociate protonated cyclo-GG, and the two lowest-energy products were the result of losses of one CO and two CO molecules. These occur from the higher-energy N-protonated tautomer, which is formed from the O-protonated tautomer by a 1,4-proton shift that has a high barrier (54.5 kcal mol(-1)) due to constraints imposed by the ring. Mechanistic schemes for four different dissociation channels, three from the N-protonated tautomer and one from the O-protonated tautomer, have been computed using density functional theory (DFT) at the B3LYP/6-311++G(d,p) level. Comparison of the potential energy surfaces for the two protonated diketopiperazines reveals the factors behind this dichotomy of fragmentation pathways. The infrared multiple-photon dissociation spectrum of the [M+H-NH3-CO](+) ion (m/z 98) from protonated cyclo-M shows this product to be an oxazole, the lowest-energy isomer. (C) 2012 Elsevier B.V. All rights reserved.

In contrast to protonated peptides, collision-induced dissociation of deprotonated peptides is known to yield abundant c- and z-type fragments, in addition to typical a-, b- and y-type ions. Here we investigate the isomeric structures of short anionic c-type peptide fragments using IR photodissociation spectroscopy. As for all N-terminal fragments of deprotonated peptides, an important structural question concerns the site of deprotonation, as no C-terminal COOH group is present in these products. Comparison of the experimental action spectra with spectra computed for several candidate structures suggests that the c(1) and c(2) ions investigated have linear peptide structures with a C-terminal -C(=O)-NH2 amide group. Competition between deprotonation on the peptide bond nitrogen atom, forming an amidate anion, and on the residue side chain, depending on its gas-phase acidity, is observed. (C) 2012 Elsevier B.V. All rights reserved.

Infrared multiple-photon dissociation (IRMPD) spectroscopy has been used to examine the gas-phase conformations of a series of short protonated polyproline ions (Pro 3 –Pro 6 ), their CID/IRMPD fragmentation pathways, and the associated fragment identities. Consistent with previous findings, and in combination with density functional theory (DFT) and MM/MD methods, a series of conformers for the protonated parent ions having their first peptide bond in the cis conformation has been identified. This conformation maximizes the solvation of the protonated N-terminus and stabilizes these compact globular-type conformations. This is in contrast to the PPI and PPII polyproline-type helices reported for larger polyproline peptides in solution. As well, this conformation leads to a unique fragmentation pattern upon collisional or multiple-photon activation. We report observation of the uncommon, but thermodynamically favored, diketopiperazine-type b 2 + fragment ion. Formation of b 2 + ions along the diketopiperazine pathway is in line with a cis configuration of the first amide linkage in the protonated parent ion. Additionally, the parent ion conformations, fragmentation pathways, and proton affinities of the resulting fragments have been related to the observed proline-effect in CID mass spectra.

Infrared ion spectroscopy using the free electron laser FELIX was applied to identify the structure of b-type peptide fragments generated by collision and IR multiple-photon induced dissociation from singly deprotonated peptides containing an asparagine residue, in particular AlaAsnAla (ANA) and AlaAlaAsnAla (AANA). IR spectra were recorded over the 800-1800 cm(-1) spectral range by multiple-photon dissociation (IRMPD) spectroscopy and have been compared with density functional theory (DFF) calculated spectra at the B3LYP/6-31++G(d,p) level for different isomeric ion structures for structural characterization. Results unambiguously show that the b(2) and b(3) fragment anions do not possess the common oxazolone or diketopiperazine structure, but involve cyclization of the asparagine side chain. Nucleophilic attack from the side chain amide nitrogen on the peptide backbone carbonyl carbon leads to the formation of cyclic succinimide structures. Deprotonation is shown to occur on the succinimide nitrogen, which delocalizes the negative charge over two adjacent carbonyl groups thus enhancing the gas-phase stability. (C) 2013 Elsevier B.V. All rights reserved.

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(center dot)) and a canonical aromatic pi (Trp(center dot+)) 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 p-radical cation was produced via CID of the ternary [Cu-II(terpy)(Trp)](center dot 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 pi 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 pi 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 similar to 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.

The gas-phase IR spectrum of the anionic chromophore of the green fluorescent protein (p-hydroxy-benzylidene-2,3-dimethylimidazolidinone, HBDI) is recorded in the 800–1800 cm−1 frequency range using the free electron laser FELIX in combination with an electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The spectrum is substantially different from IR spectra of the anion recorded previously in solution, which were found to be difficult to interpret on the basis of electronic structure calculations involving the polarisable continuum model (PCM) method. In contrast, the IR spectrum of the isolated anion recorded here matches favourably with its DFT calculated counterpart if diffuse functions are included in the basis set. IR photo-fragmentation of the HBDI anion proceeds via loss of a methyl radical (CH3) resulting in an odd-electron product anion. The IR spectrum of this radical anion photoproduct is also recorded, which indicates that the radical site resides on the imidazolinone nitrogen atom where the methyl group is detached.

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.

Andrii Piatkivskyi, Sandra Osburn, Kendall Jaderberg, Josipa Grzetic, Jeffrey D. Steill, Jos Oomens, Junfang Zhao, Justin Kai-Chi Lau, Udo H. Verkerk, Alan C. Hopkinson, K. W. Michael Siu, Victor Ryzhov Department of Chemistry and Biochemistry, and Center for Biochemical and Biophysical Studies, Northern Illinois University, DeKalb, IL 60115, USA FOM Institute for Plasma Physics, Rijnhuizen 14, 3439 MN Nieuwegein, The Netherlands University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands Institute for Molecules and Materials (IMM), FELIX facility, Radboud University Nijmegen, Heyendaalseweg 135, 6525AJ Nijmegen, The Netherlands Department of Chemistry and Centre for Research in Mass Spectrometry, York University, Toronto, ON, Canada Sandia National Laboratories, Livermore, CA 94550-0969, USA

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 material The online version of this article (doi:10.1007/s13361-016-1462-5) contains supplementary material, which is available to authorized users.