An unprecedented method of producing molecular radical cations of oligopeptides in the gas phase has been discovered. Electrospraying a methanolic mixture of a Cu(II)-amine complex, e.g., Cu II (dien)(NO 3 ) 2 (where dien ) diethylenetriamine), and an oligopeptide (M) yields the [Cu II (dien)M] •2+ ion, whose collision-induced dissociation (CID) produces [Cu I (dien)] + and M •+ , the molecular cation of the oligopeptide. Abundant M •+ is apparent when the oligopeptide contains both a tyrosyl and a basic residuesarginyl, lysyl, or histidyl. These structural requirements are similar to those in the metalloradical enzyme process in photosystem II. Tandem mass spectrometry of M •+ produces fragment ions that are both common to and also different from [M + H] + . The fragmentation chemistry of M •+ and of its products appear to be radical driven.Protein radicals have generated a lot of recent interest because of their unusual role in catalyzing a number of important reactions, 1 including the oxidation of water to oxygen for use in a photosynthesis system in plants and algae. 1a,b A common theme in these protein radicals is that they are synthesized posttranslationally and their formation involves metallo cofactors located either adjacent to the amino acid residue being oxidized or on a second subunit or activating enzyme that participates in the oxidation. 1 Frequent radical sites are located on the glycyl, tyrosyl, and tryptophanyl residues; the structure of the glycyl radical in pyruvate formate lyase has been found to be planar and maintain a gas-phase-like structure, despite being embedded in the protein. 1d Here we report results of a serendipitous discovery of an unprecedented route for generating molecular radical cations of oligopeptides in the gas phase. Some of the conditions under which these oligopeptide radical cations are generated bear a resemblance to those in vivo for protein radicals. It is noteworthy that this discovery centers on molecular radical cations as opposed to the more frequently encountered radical ions produced from protonated peptides capturing an electron 2 and metal-bearing peptide ternary complexes. 3 Although electron ionization (EI) is the most widely used method in mass spectrometry to generate radical cations, it is not amenable to oligopeptides because of their low volatility. Very few dipeptides and, as far as we know, only one tripeptide have been successfully ionized in this manner; their mass spectra were rich and contained a wealth of sequencing information. 4 We are reporting herein that electrospraying a methanolic mixture of a Cu(II)-amine complex, e.g., Cu II (dien)(NO 3 ) 2 (where dien ) diethylenetriamine), and an oligopeptide (M) yields the [Cu II -(dien)M] •2+ ion, whose collision-induced dissociation (CID) produces [Cu I (dien)] + and M •+ , the odd-electron (OE) molecular cation of the oligopeptide. Abundant M •+ is apparent only when the oligopeptide contains a tyrosyl and a basic residuesarginyl, lysyl, or histidyl. Figure 1 shows the product ion spectra of...
The fragmentation mechanisms of protonated triglycine and its first-generation dissociation products have been investigated using a combination of density functional theory calculations and threshold collision-induced dissociation experiments. The activation barrier measured for the fragmentation of protonated triglycine to the b(2) ion and glycine is in good agreement with a calculated barrier at the B3LYP/6-31++G(d,p) level of theory reported earlier [Rodriquez, C. F. et al. J. Am. Chem. Soc. 2001, 123, 3006-3012]. The b(2) ion fragments to the a(2) ion via a transition state structure that is best described as acylium-like. Contrary to what is commonly assumed, the lowest energy structure of the a(2) ion is not an iminium ion, but a cyclic, protonated 4-imidazolidone. Furthermore, fragmentation of the b(2) to the a(1) ion proceeds not via a mechanism that results in HNCO and H(2)C=C=O as byproducts, as have been postulated, but via a transition state that contains an incipient a(1) ion and an incipient carbene. The fragmentation of a(2) to a(1) proceeds via a transition state structure that contains the a(1) ion, CO and an imine as incipient components.
Tandem mass spectrometry performed on a pool of 18 oligopeptides shows that the product ion spectra of argentinated peptides, the [bn + OH + Ag]+ ions and the [yn - H + Ag]+ ions bearing identical sequences are virtually identical. These observations suggest strongly that these ions have identical structures in the gas phase. The structures of argentinated glycine, glycylglycine, and glycylglycylglycine were calculated using density functional theory (DFT) at the B3LYP/DZVP level of theory; they were independently confirmed using HF/LANL2DZ. For argentinated glycylglycylglycine, the most stable structure is one in which Ag+ is tetracoordinate and attached to the amino nitrogen and the three carbonyl oxygen atoms. Mechanisms are proposed for the fragmentation of this structure to the [b2 + OH + Ag]+ and the [Y2 - H + Ag]+ ions that are consistent with all experimental observations and known calculated structures and energetics. The structures of the [b2 - H + Ag]+ and the [a2 - H + Ag]+ ions of glycylglycylglycine were also calculated using DFT. These results confirm earlier suggestions that the [b2 - H + Ag]+ ion is an argentinated oxazolone and the [a2 - H + Ag]+ an argentinated immonium ion.
Density functional calculations at B3LYP/DZVP were used to obtain structural information, relative free energies of di †erent isomers and binding energies for the following reaction in the gas phase : Mẁ here M \ Ag or Cu and n \ 0È2. For the complexes with Cu`, ] (glycyl)n glycine ] MÈ(glycyl) n glycine`, optimizations were also performed at B3LYP/6È31&&G(d,p) and single-point calculations at MP2(fc)/6È311&&G(2df,2p)//B3LYP/DZVP. The calculated binding energies for the Cu`complexes are all higher than those of the structurally similar Ag`ions. These calculated binding energy di †erences become larger as the size of the ligand increases. For all the Cu`complexes examined, the coordination number of the copper ion does not exceed two, whereas for the silver complexes tri-and tetracoordinate Ag`structures are calculated to be at low energy minima. SigniÐcant structural and relative free energy di †erences occur between the lowest energy " zwitterionic Ï forms of the complexes. MÈ(glycyl) n glycineÌ
The binding energies at 0 K of sodium and silver ions to ammonia, methylamine, ethylamine, acetonitrile, and benzonitrile were determined using threshold collision-induced dissociation (CID) and molecular orbital calculations at the ab initio and density functional theory levels. There is good agreement between experimental and calculated binding energies. For the five ligands, threshold CID/CCSD(t)(fu)/6-311++G(2df,p)//MP2(fu)/6-311++G(d,p) Na+ binding energies are the following: ammonia, 25.6 ± 2.8/24.8; methylamine, 27.0 ± 1.4/25.9; ethylamine, 27.7 ± 2.3/27.1; acetonitrile, 30.0 ± 2.3/30.3; and benzonitrile, 32.7 ± 1.4/35.0 (B3LYP/6-311++G(d,p)//B3LYP/6-311++G(d,p)) kcal/mol. Threshold CID and B3LYP/DZVP Ag+ binding energies are the following: ammonia, 40.6 ± 3.0/38.9; methylamine, 41.5 ± 2.3/41.1; ethylamine, 42.9 ± 1.4/43.2; acetonitrile, 40.8 ± 2.0/39.3; and benzonitrile, 41.5 ± 2.8/43.1 kcal/mol. Wherever comparisons with literature data are possible, the Na+ binding energies determined in this study are in good agreement with established data. For Ag+ binding energies, agreement with the few published theoretical values is not as good. A comparison of Na+ and Ag+ binding energies for the five N-containing ligands in this study and those for water, methanol, and ethanol published earlier (El Aribi, H.; Shoeib, T.; Ling, Y.; Rodriquez, C. F.; Hopkinson, A. C.; Siu, K. W. M. J. Phys. Chem. A 2002, 106, 2908−2914) shows that for every ligand the Ag+ binding energy is higher than the Na+ binding energy. As a group, the amines exhibit the largest differences between Ag+ and Na+ binding energies, followed by the nitriles; the alcohols exhibit the smallest differences. These results are in line with previous observations that Ag+ prefers binding with nitrogen to binding with oxygen.
The binding enthalpies at 0 K of the silver ion to water, methanol, ethanol, diethyl ether, and acetone were calculated using density functional theory (DFT) using the hybrid B3LYP level of theory with the DZVP basis set; they were also measured using the threshold collision-induced dissociation (CID) method. There is good agreement between the two sets of data. For the five ligands, the DFT/threshold CID values are: water, 28.1/31.6 ± 2.5; methanol, 30.1/33.0 ± 3.7; ethanol, 32.0/33.9 ± 3.5; diethyl ether, 33.3/33.2 ± 1.5; and acetone, 36.2/38.0 ± 1.4 kcal/mol. The average of the absolute differences between the DFT and threshold CID results is 2.0 kcal/mol, a value smaller than the average experimental uncertainty of 2.5 kcal/mol. For identical ligands, the silver ion binding energies are lower than the lithium ion binding energies, but higher than the sodium ion binding energies.
Molecular radical cations have proven to be difficult to generate from aliphatic peptides under electrospray ionization mass spectrometry (ESI-MS) conditions. For a family of small aliphatic peptides GGX, where X = G, A, P, I, L and V, these cations have been generated by electrospraying a mixture of Cu.2+, 12-crown-4 and GGX in methanol/water. GGX.+ is readily formed from the collision-induced dissociation (CID) of [CuII(12-crown-4)(GGX)].2+. The formation of these aliphatic peptide radical ions from these complexes, in cases where it is not possible from the corresponding complexes involving a series of amine ligands instead of 12-crown-4, is likely due to the second ionization energy of the [CuI(12-crown-4)(GGX)]+ complex being higher than that of the corresponding [CuI(amine)(GGX)]+ complex. Using these 12-crown-4 complexes, GGI can be differentiated from the isomeric GGL by comparing the CID spectra of their [a3 + H].+ ions.
The conformers of gaseous bradykinin, BK, (Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)) and its protonated forms, [BK + H](+), [BK + 2H](2+), and [BK + 3H](3+), were examined theoretically using a combination of the Merck molecular force field, Hartree-Fock, and density functional theory. Neutral BK, [BK + H](+), and [BK + 2H](2+) exist in zwitterionic forms that are stabilized by internal solvation and have compact structures; [BK + 3H](3+) differs by the absence of a salt bridge and adopts an elongated form. The common structural feature in all four BK species is a beta-turn in the Ser(6)-Pro(7)-Phe(8)-Arg(9) sequence. The gas-phase basicity of [BK + H](+) estimated from the calculated protonation energy is in accord with published experimental basicity; population-weighted collision cross-sections of the three ionic forms are in agreement with experimental cross-sections in the literature.
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