Sequential hydration of small protonated peptides

The sequential addition of water molecules to a series of small protonated peptides was studied by equilibrium experiments using electrospray ionization combined with drift cell techniques. The experimental data were compared to theoretical structures of selected hydrated species obtained by molecular mechanics simulations. The sequential water binding energies were measured to be of the order of 7-15 kcal/mol, with the largest values for the first water molecule adding to either a small nonarginine containing peptide (e.g., protonated dialanine) or to a larger peptide in a high charge state (e.g., triply protonated neurotensin). General trends are (a) that the first water molecules are more strongly bound than the following water molecules, (b) that very small peptides (2-3 residues) bind the first few water molecules more strongly than larger peptides, (c) that the first few water molecules bind more strongly to higher charge states than to lower charge states, and (d) that water binds less strongly to a protonated guanidino group (arginine containing peptides) than to a protonated amino group. Experimental differential entropies of hydration were found to be of the order of -20 cal/mol/K although values vary from system to system. At constant experimental conditions the number of water molecules adding to any peptide ion is strongly dependent on the peptide charge state (with higher charge states adding proportionally more water molecules) and only weakly dependent on the choice of peptide. For small peptides molecular mechanics calculations indicate that the first few water molecules add preferentially to the site of protonation until a complete solvation shell is formed around the charge. Subsequent water molecules add either to water molecules of the first solvation shell or add to charge remote functional groups of the peptide. In larger peptides, charge remote sites generally compete more effectively with charge proximate sites even for the first few water molecules.     

Ion–molecule reactions of fragment ions derived from protonated allyl methyl ether

Unusual behaviour has been noted for allyl methyl ether (1) chemically ionized in a high-pressure ion source. Tandem mass spectrometry indicates the formation of methylcyclopentadienyl and methoxy-1-propenylcarbenium ions (d, m/z 81 and e, m/z 85). The origin of these unexpected ions has been elucidated using conventional and Fourier transform ion cyclotron resonance experiments: primary fragment ions derived from protonated 1 (allyl ions a and methoxymethyl cations b) generate collision complexes with neutral 1, giving rise to the ions d and e, respectively, after methanol elimination.     

Cyclization and rearrangement reactions of a(n) fragment ions of protonated peptides

a(n) ions are frequently formed in collision-induced dissociation (CID) of protonated peptides in tandem mass spectrometry (MS/MS) based sequencing experiments. These ions have generally been assumed to exist as immonium derivatives (-HN(+)═CHR). Using a quadrupole ion trap mass spectrometer, MS/MS experiments have been performed and the structure of a(n) ions formed from oligoglycines was probed by infrared spectroscopy. The structure and isomerization reactions of the same ions were studied using density functional theory. Overall, theory and infrared spectroscopy provide compelling evidence that a(n) ions undergo cyclization and/or rearrangement reactions, and the resulting structure(s) observed under our experimental conditions depends on the size (n). The a(2) ion (GG sequence) undergoes cyclization to form a 5-membered ring isomer. The a(3) ion (GGG sequence) undergoes cyclization initiated by nucleophilic attack of the carbonyl oxygen of the N-terminal glycine residue on the carbon center of the C-terminal immonium group forming a 7-membered ring isomer. The barrier to this reaction is comparatively low at 10.5 kcal mol(-1), and the resulting cyclic isomer (-5.4 kcal mol(-1)) is more energetically favorable than the linear form. The a(4) ion with the GGGG sequence undergoes head-to-tail cyclization via nucleophilic attack of the N-terminal amino group on the carbon center of the C-terminal immonium ion, forming an 11-membered macroring which contains a secondary amine and three trans amide bonds. Then an intermolecular proton transfer isomerizes the initially formed secondary amine moiety (-CH(2)-NH(2)(+)-CH(2)-NH-CO-) to form a new -CH(2)-NH-CH(2)-NH(2)(+)-CO- form. This structure is readily cleaved at the -CH(2)-NH(2)(+)- bond, leading to opening of the macrocycle and formation of a rearranged linear isomer with the H(2)C═NH(+)-CH(2)- moiety at the N terminus and the -CO-NH(2) amide bond at the C terminus. This rearranged linear structure is much more energetically favorable (-14.0 kcal mol(-1)) than the initially formed imine-protonated linear a(4) ion structure. Furthermore, the barriers to these cyclization and ring-opening reactions are low (8-11 kcal mol(-1)), allowing facile formation of the rearranged linear species in the mass spectrometer. This finding is not limited to 'simple' glycine-containing systems, as evidenced by the IRMPD spectrum of the a(4) ion generated from protonated AAAAA, which shows a stronger tendency toward formation of the energetically favorable (-12.3 kcal mol(-1)) rearranged linear structure with the MeHC═NH(+)-CHMe- moiety at the N terminus and the -CO-NH(2) amide bond at the C terminus. Our results indicate that one needs to consider a complex variety of cyclization and rearrangement reactions in order to decipher the structure and fragmentation pathways of peptide a(n) ions. The implications this potentially has for peptide sequencing are also discussed.     

Doubly charged fragment ions in the field ionization mass spectra of alkylbenzenes

It is shown that the radical [C₆H₅C_mH_2m]²⁺ fragment ions found in the field ionization mass spectra of alkylbenzenes are formed via a different adsorption state of the singly charged species than in the case of the formation of [M]²⁺ molecular ions. It is further demonstrated that the primary fragmentation of molecules by the cleavage of C? C bonds results not only from decompositions of molecular ions in the gas phase but also from surface reactions.     

Doubly charged ion clusters

The electron impact ionization of hydrogen-bonded clusters of water and ammonia results in the formation of stable doubly charged ions only when the cluster size exceeds a critical value. The observed minimum cluster size for water is (H₂O)₃₅H₂²⁺ and for ammonia is (NH₃)₅₁H₂⁺. From a knowledge of the relative hydrogen-bond strenghts, it has been possible to account for the size difference between these ions and to qualitatively discuss the locations of the positive charges within each cluster.     

Doubly protonated benzene in the gas phase

Structural aspects and the unimolecular fragmentations of doubly protonated benzene are studied by means of tandem-mass spectrometry. The corresponding dications are generated by electron ionization (EI) of 1,3- and 1,4-cyclohexadienes, respectively. It is suggested that EI of 1,3-cyclohexadiene leads to the singlet state of doubly protonated benzene, whereas EI of 1,4-cyclohexadiene yields a mixture of singlet and triplet states. Unimolecular fragmentation of doubly protonated benzene exclusively proceeds via dehydrogenation leading to the benzene dication. The proton affinities (PAs) of protonated benzene amount to PA(C(6)H(7)(+))(meta) = 1.9 +/- 0.3 eV for protonation taking place at the meta-position, PA(C(6)H(7)(+))(ortho) = 1.5 +/- 0.2 eV, and PA(C(6)H(7)(+))(para) = 0.9 +/- 0.2 eV, respectively. Various facets of the experiments are compared with density functional theory calculations and generally good agreement is found.     

Fragmentation characteristics of b(n) (n=2-15) ions from protonated peptides

The fragmentation reactions of protonated oligoalanines (trialanine, tetraalanine and pentaalanine) and the fragments present in the electrospray ionization (ESI) mass spectrum of polyalanine have been studied by collisionally activated dissociation (CAD) mass spectrometry (MS(2) and MS(3) experiments). The MS(n) experiments provided strong evidence that the m/z 71n+1 ion series in the ESI mass spectrum of polyalanine is a b(n) series. These ions are formed via the b(n) -y(m) pathway of amide bond cleavage, which results in the formation of a proton-bound complex of an oxazolone and a peptide/amino acid. Also, the MS(2) spectra of the b(n) series from polyalanine revealed that the chain length of b(n) ions influences significantly the dissociations taking place. For example, b(n) ions start losing H(2)O at n ≥5 and the losses of CO and CO+NH(3) decrease in intensity from b(2) to b(15). The elimination of H(2)O+NH(3) and the elimination of 61 mass (HN=C=O+H(2)O) commence with b(6); their abundances initially increase up to ~ b(8)-b(9) and then gradually decrease until b(15) (largest fragment studied). The tandem mass spectrometry experiments help to elucidate the dissociation mechanisms of the observed structures of biopolymer fragments.     

Photochemical generation of nitrenium ions from protonated 1,1-diarylhydrazines

[reaction: see text] Laser flash photolysis experiments, chemical trapping studies, and time-dependent density functional theory calculations demonstrate that photolysis of protonated 1,1-diarylhydrazines generates N,N-diarylnitrenium ions.     

Bioactive peptides derived from food

As interest in the ability of functional foods to impact on human health has grown over the past decade, so has the volume of knowledge detailing the beneficial roles of food-derived bioactive peptides. Bioactive peptides from both plant and animal proteins have been discovered, with to date, by far the most being isolated from milk-based products. A wide range of activities has been described, including antimicrobial and antifungal properties, blood pressure-lowering effects, cholesterol-lowering ability, antithrombotic effects, enhancement of mineral absorption, immunomodulatory effects, and localized effects on the gut. Although there is still considerable research to be performed in the area of food-derived bioactive peptides, it is clear that the generation of bioactive peptides from dietary proteins during the normal digestive process is of importance. Therefore, it will become necessary when determining dietary protein quality to consider the potential effects of latent bioactive peptides that are released during digestion of the protein.     

Evaporation from water clusters containing singly charged ions

Molecular dynamics simulations were used to study the evaporation from water clusters containing either Cl(-), H(2)PO(4)(-), Na(+) or NH(4)(+) ions. The simulations ranged between 10 and 500 ns, and were performed in vacuum starting at 275 K. A number of different models were used including polarizable models. The clusters contain 216 or 512 molecules, 0, 4 or 8 of which were ions. The ions with hydrogen bonding properties do not affect evaporation, even though the phosphate ions have a pronounced ion-ion structure and tend to be inside the cluster whereas ammonium shows little ion-ion structure and has a distribution within the cluster similar to that of the water molecules. Since the individual ion-water interactions are much stronger in the case of Na(+)-water and Cl(-)-water clusters, evaporation is somewhat slower for clusters containing these ions. It seems therefore that the main determinant of the evaporation rate in ion-water clusters is the strength of the interaction. Fission of droplets that contain more ions than allowed according to the Rayleigh limit seems to occur more rapidly in clusters containing ammonium and sodium ions.     

Skeletal rearrangement for water loss from molecular protonated ions of t-butoxycyclohexane

Isotopic labelling and chemical substitution support the proposition that the skeletal rearrangement for water loss from molecular protonated ions of t-butoxycyclohexane involves competition between three reaction pathways. The principal reaction pathway (83%) involves migration of the t-butyl group to the 2-(6-) position of the cyclohexyl ring with reciprocal hydrogen transfer. A second reaction pathway (12%) involves ring contraction followed by reciprocal exchange of the t-butyl group with the 2-(5-) hydrogen atom of the nascent cyclopentyl ring. The third reaction pathway (5%) involves rearrangement of a proton-bound complex to permit ipso attack by isobutene. Stereospecific substitutions indicate that the principal reaction pathway is susceptible to 1,3-diaxial interactions.     

Doubly charged ion mass spectra of organophosphorus compounds

Doubly charged ion mass spectra have been obtained for 11 organophosphorus compounds. Methane has been used as a target gas to increase the probability of single electron transfer collisions in the first field-free region of an Hitachi RMU-7L mass spectrometer. In general, the spectra of organophosphorus compounds do not exhibit molecular ions but are dominated by fragment ions, many of which must be formed by rearrangement processes. A geometry-optimized self-consistent field molecular orbital method has been employed to compute energies and structural parameters for prominent ions. In addition, a diabatic curve crossing model has been used to examine the single electron transfer reactions responsible for intense ions in the doubly charged ion mass spectra. Appearance energies measured for ions prominent in the 2E spectra of organophosphorus compounds have ranged from 23 to 38 eV.     

Fragmentation of protonated ions of peptides containing cysteine,cysteine sulfinic acid,and cysteine sulfonic acid

The oxidation of the sulfhydryl group in cysteine to sulfenic acid, sulfinic acid, and sulfonic acid in proteins is important in a number of enzymatic processes. In this study we examined the fragmentation of four peptides containing cysteine, cysteine sulfinic acid (Cys-SO(2)H), and cysteine sulfonic acid (Cys-SO(3)H) in an ion-trap mass spectrometer. Our results show that the presence of a Cys-SO(2)H in a peptide leads to preferential cleavage of the amide bond at the C-terminal side of the oxidized cysteine residue. The results are important for the determination of the site of the cysteine oxidation and might be useful for the sequencing of cysteine-containing peptides.     

Multiply charged ions in field desorption mass spectra of peptides containing basic amino acids

Multiply charged ions up to [M+4]⁴⁺ were observed in the field desorption mass spectra of five different peptides. The relationship between the charge-multiplicity and the number of basic sidechains is discussed.     

Apparent inhibition by arginine of macrocyclic <Emphasis Type=Italic>b</Emphasis> ion formation from singly charged protonated peptides

There is now strong evidence for the existence of macrocyclic isomers of b_n⁺ ions, the formation and subsequent opening of which can lead to loss of sequence information from protonated peptides in multiple-stage tandem mass spectrometry experiments. In this study, the fragmentation patterns of protonated YARFLG and permuted isomers of the model peptide were investigated by collision-induced dissociation. Of interest was the potential influence of the arginine residue, and its position in the peptide sequence, on formation of the presumed macrocyclic b₅ ion isomer and potential loss of sequence information. We find that regardless of the sequence position (either internal or at the N- or C-terminus), only direct sequence ions or ions directly related to fragmentation of the arginine side chain are observed.     

Evaporation kinetics of tetraalkylammonium ions from charged formamide drops

The rate of ion evaporation from the surface of electrically charged liquid drops may be inferred from observations of the minimum drop charge q present on drops of a given radius R. This critical relation q(R) is measured here from the fossil solid residues left by the drops after complete solvent evaporation. We obtain mobility distributions of singly charged clusters formed by charge-reduced electrosprays of tetra-n-alkylammonium salts (C(n)()H(2)(n)()(+1))(4)N(+) (n = 2-10) dissolved in formamide. These distributions exhibit modulated structures, with each wave being associated with an initial charge state of the clusters prior to charge reduction, from which critical q(R) relations follow. For n from 4 to 7, the behavior is weakly dependent on the length of the alkyl chain. Above n = 7, there is a marked increase in solvation energy of the alkylammonium ions, but drop curvature effects contribute a compensating reduction of the energy barrier for ionization. This curvature effect increases monotonically with n and is probably associated with surface activity. Few clear modulations are seen for n < 3, perhaps because of the decreased role of surface activity in transferring solute into very small drops during the Coulombic breakup of larger drops. For this reason, extension of this technique to small inorganic salts is problematic.