Search

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., Mass spectrometry is a leading method used for sequencing peptides and proteins by fragmentation followed by analysis of the sequence fragments. Here, the authors use infrared spectroscopy to characterize the structures of peptide fragments formed during electron transfer dissociation.

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.

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.

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.