Hydration of potassiated amino acids in the gas phase

The thermochemistry of stepwise hydration of several potassiated amino acids was studied by measuring the gas-phase equilibria, AAK(+)(H(2)O)(n-1) + H(2)O = AAK(+)(H(2)O)(n) (AA = Gly, AL, Val, Met, Pro, and Phe), using a high-pressure mass spectrometer. The AAK(+) ions were obtained by electrospray and the equilibrium constants K(n-1,n) were measured in a pulsed reaction chamber at 10 mbar bath gas, N(2), containing a known partial pressure of water vapor. Determination of the equilibrium constants at different temperatures was used to obtain the DeltaH(n)(o), DeltaS(n)(o), and DeltaG(n)(o) values. The results indicate that the water binding energy in AAK(+)(H(2)O) decreases as the K(+) affinity to AA increases. This trend in binding energies is explained in terms of changes in the side-chain substituent, which delocalize the positive charge from K(+) to AA in AAK(+) complexes, varying the AAK(+)-H(2)O electrostatic interaction.     

Vibrational signatures of protonated, phosphorylated amino acids in the gas phase

Structural characterization of protonated phosphorylated serine, threonine, and tyrosine was performed using mid-infrared multiple photon dissociation (IRMPD) spectroscopy and density functional theory (DFT) calculations. The ions were generated and analyzed by an external electrospray source coupled to a Paul ion-trap type mass spectrometer. Their fragmentation was induced by the resonant absorption of multiple photons from a tunable free electron laser (FEL) beam. IRMPD spectra were recorded in the 900-1850 cm(-1) energy range and compared to the corresponding computed IR spectra. On the basis of the frequency and intensity of two independent bands in the 900-1400 cm(-1) energy range, it is possible to identify the phosphorylated residue. IRMPD spectra for a 12-residue fragment of stathmin in its phosphorylated and nonphosphorylated forms were also recorded in the 800-1400 cm(-1) energy range. The lack of spectral congestion in the 900-1300 cm(-1) region makes their distinction facile. Our results show that IRMPD spectroscopy may became a valuable tool for structural characterization of small phosphorylated peptides.     

Infrared spectroscopy of hydrated amino acids in the gas phase: protonated and lithiated valine

We report here infrared spectra of protonated and lithiated valine with varying degrees of hydration in the gas phase and interpret them with the help of DFT calculations at the B3LYP/6-31++G** level. In both the protonated and lithiated species our results clearly indicate that the solvation process is driven first by solvation of the charge site and subsequently by formation of a second solvation shell. The infrared spectra of Val x Li+ (H2O)4 and Val x H+ (H2O)4 are strikingly similar in the region of the spectrum corresponding to hydrogen-bonded stretches of donor water molecules, suggesting that in both cases similar extended water structures are formed once the charge site is solvated. In the case of the lithiated species, our spectra are consistent with a conformation change of the amino acid backbone from syn to anti accompanied by a change in the lithium binding from a NO coordination to OO coordination configuration upon addition of the third water molecule. This change in the mode of metal ion binding was also observed previously by Williams and Lemoff [J. Am. Soc. Mass Spectrom. 2004, 15, 1014-1024] using blackbody infrared radiative dissociation (BIRD). In contrast to the zwitterion formation inferred from results of the BIRD experiments upon addition of a third water molecule, our spectra, which are a more direct probe of structure, show no evidence for zwitterion formation with the addition of up to four water molecules.     

Resolution of sulphur-containing amino acids by chiral phase gas chromatography

Summary Methyl esters of the pentafluoropropionyl-amino acid derivatives of the tetrafunctional, sulphur-bridged, stereoisomeric lanthionines, cystathionines and -methyl-lanthionines were resolved on glass capillaries coated with the chiral stationary phase N-propionyl-L-valine-N-tert-butylamide-polysiloxane (Chirasil-Val) within 35min. Interestingly, L-cystathionine elutes before its D-enantiomer in contrast to the usual order of emergence on an L-phase. The method was applied to the polypeptide antibiotic nisin, which contains mesolanthionine and 2S,3S,6R-3-methyl-lanthionine.N-Pentafluoropropionyl-S-alkylthiocysteine methyl esters (R=methyl, ethyl, n- and iso-propyl, n- and sec-butyl, n-octyl, neo-pentyl, cyclohexyl-, benzyl-, tolyl-) were separated on Chirasil-Val within 30min. The identity of all derivatives was shown by combined gas chromatography-mass spectrometry.     

Reactions of charged phenyl radicals with aliphatic amino acids in the gas phase

Gas-phase reactivity of five differently substituted positively charged phenyl radicals was examined toward six amino acids by using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR). The reactivity of the radicals studied was determined by the electrophilicity of the radical, which can be characterized by the radical's electron affinity (EA). The larger the electron affinity of the radical, the higher the overall reaction rate. In addition to the expected H-atom abstraction, several unprecedented reaction pathways were observed, including NH2 abstraction, SH abstraction, and SCH3 abstraction. These reaction pathways dominate for the most electrophilic radicals, and they may not follow radical but rather nucleophilic addition-elimination mechanisms. Hydrogen abstraction from glycine was also investigated theoretically. The results indicate that hydrogen abstraction from alphaC of glycine is both kinetically and thermodynamically favored over the NH2 group. The ordering of transition state energies for hydrogen abstraction from the alphaC and NH2 groups was found to reflect the radicals' EA ordering.     

Hydration energies of deprotonated amino acids from gas phase equilibria measurements

Singly hydrated clusters of deprotonated amino acids were studied using an electrospray high-pressure mass spectrometer equipped with a pulsed ion-beam reaction chamber. Thermochemical data, DeltaH(o), DeltaS(o), and DeltaG(o), for the hydration reaction [AA - H](-) + H(2)O = [AA - H](-).(H(2)O) were obtained from gas-phase equilibria determinations for AA = Gly, Ala, Val, Pro, Phe, Lys, Met, Trp, Gln, Arg, and Asp. The hydration free-energy changes are found to depend significantly on the side-chain substituents. The water binding energy in [AA - H](-).(H(2)O) increases with the gas-phase acidity of AA. The anionic hydrogen bond strengths in [AA - H](-).(H(2)O) are compared with those of the cationic bonds in the corresponding AAH(+).(H(2)O) systems.     

Fingerprint vibrational spectra of protonated methyl esters of amino acids in the gas phase

Infrared spectra of the protonated monomers of glycine, alanine, valine, and leucine methyl esters are presented. These protonated species are generated in the gas phase via matrix assisted laser desorption ionization (MALDI) within the cell of a Fourier transform ion cyclotron resonance spectrometer (FTICR) where they are subsequently mass selected as the only species trapped in the FTICR cell. Alternatively, they have also been generated by electrospray ionization and transferred to a Paul ion-trap mass spectrometer where they are similarly isolated. In both cases IR spectra are then derived from the frequency dependence of the infrared multiple photon dissociation (IRMPD) in the mid-infrared region (1000-2200 cm(-1)), using the free electron laser facility Centre de Laser Infrarouge d'Orsay (CLIO). IR bands are assigned by comparison with the calculated vibrational spectra of the lowest energy isomers using density functional theory (DFT) calculations. There is in general good agreement between experimental IRMPD spectra and calculated IR absorption spectra for the lowest energy conformer which provides evidence for conformational preferences. The two different approaches to ion generation and trapping yield IRMPD spectra that are in excellent agreement.     

Selective transport of amino acids into the gas phase: driving forces for amino acid solubilization in gas-phase reverse micelles

We report a study on encapsulation of various amino acids into gas-phase sodium bis(2-ethylhexyl) sulfosuccinate (NaAOT) reverse micelles, using electrospray ionization guided-ion-beam tandem mass spectrometry. Collision-induced dissociation of mass-selected reverse micellar ions with Xe was performed to probe structures of gas-phase micellar assemblies, identify solute-surfactant interactions, and determine preferential incorporation sites of amino acids. Integration into gas-phase reverse micelles depends upon amino acid hydrophobicity and charge state. For examples, glycine and protonated amino acids (such as protonated tryptophan) are encapsulated within the micellar core via electrostatic interactions; while neutral tryptophan is adsorbed in the surfactant layer. As verified using model polar hydrophobic compounds, the hydrophobic effect and solute-interface hydrogen-bonding do not provide sufficient driving force needed for interfacial solubilization of neutral tryptophan. Neutral tryptophan, with a zwitterionic structure, is intercalated at the micellar interface between surfactant molecules through complementary effects of electrostatic interactions between tryptophan backbone and AOT polar heads, and hydrophobic interactions between tryptophan side chain and AOT alkyl tails. Protonation of tryptophan could significantly improve its incorporation capacity into gas-phase reverse micelles, and displace its incorporation site from the micellar interfacial zone to the core; protonation of glycine, on the other hand, has little effect on its encapsulation capacity. Another interesting observation is that amino acids of different isoelectric points could be selectively encapsulated into, and transported by, reverse micelles from solution to the gas phase, based upon their competition for protonation and subsequent encapsulation within the micellar core.     

Complexation of Al(III) by aromatic amino acids in the gas phase

The coordination properties of three natural aromatic amino acids (AAAs)-phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp)-to AlIII are studied in this work, devoting special attention to the role of the aromatic side chain. A comparison with aluminum(III)-alanine complexes is also presented. The polarizability arising from the ring has been seen to be a key factor in the stability of the complexes, with the order being Trp-AlIII > Tyr-AlIII > Phe-AlIII, starting from the most stable one. Cation-pi interactions between the metal and the aromatic ring are present in the lowest energy conformers, especially for Trp, which seems to be very well suited for these kinds of interactions, occurring with both the six- and five-membered rings of the indole side chain. The most stable coordination mode for the three AAAs is found to be tricoordinated with the N and O of the backbone chain and the aromatic ring, as was found theoretically and experimentally for other metals.     

Reactions of gas phase amino acids with the prevailing radicals in biomass thermal treatments

Amino acids, N-containing compounds, hold a significant importance in various field. Within the biomass energy sector, amino acids constitute a large fraction of the biomass's nitrogen content. As such, it is essential to comprehend their combustion chemistry; most specifically their biomolecular interactions with governing radicals in the pyrolytic and combustion media that prevail during thermal utilization of biomass. Herein, we have employed quantum chemical calculations and reaction rate theory to investigate reactions of a selected set of amino acids with H, CH₃, NH₂, OH, HO₂, and HS radicals. Thermo-kinetic calculations have been performed to determine the rates of hydrogen abstraction by these six radicals across all possible reaction channels for three specific amino acids: alanine, cysteine, and methionine. The investigation of other amino acids like glycine, threonine, and other models have been carried out for α-C positions as the most probable abstractable sites. The study also examines the individual effects of different substituents (COOH, NH₂, HS, and CH₂) and uncovers significant insights. Notably, the presence of the COOH group introduces polar effects that counterintuitively deactivate the thermodynamically favored α-abstraction pathway. Presented thermo-kinetic values are anticipated to complement existing biomass kinetic models and to improve current understanding of chemical events that participate in the complex nitrogen transformation reactions in biomass.     

On the question of salt bridges of cationized amino acids in the gas phase: glycine and arginine

The geometrical shapes of the sodiated and cesiated amino acids glycine and arginine were probed in the gas phase by using the ion mobility based ion chromatography method. The data were compared to those obtained for alkali cationized methyl esters and for all the protonated species. Molecular mechanics, semiempirical, and ab initio/density functional theory (DFT) calculations were carried out to generate model structures for comparison with experiment and to determine the relative energies of different structures. For alkali cationized glycine the experimental cross sections agreed with charge solvation structures which were found by calculation to be the most stable forms as well. Both experiment and theory indicated that sodium is solvated by both the amino and the carbonyl groups, while cesium is solvated by one or both oxygen(s) of the carboxyl group. Alkali cationized arginine was found to form a salt bridge structure. The carboxylate group is stabilized by both the charged guanidinium group and the alkali ion. High level (6-311++G7 and DZVP) ab initio/DFT calculations carried out on sodiated and rubidiated N amidinoglycine, which contains a guanidino group and which was used as a model for the larger arginine molecule, indicated that the salt bridge structures are ∼10 kcal/mol more stable than the charge solvation forms for both alkali ions. The structure of protonated arginine, i.e. salt bridge or charge solvation, could not be unambiguously determined.     

Hydrogen-deuterium exchange and selective labeling of deprotonated amino acids and peptides in the gas phase

Hydrogen-deuterium exchange reactions of deprotonated amino acids and small peptides were studied. Selective labeling can be carried out at the alpha-amino group of lysine (2 of 4 labile hydrogens undergo exchange with CF3CH2OD) and the guanidine side chain of arginine (3 of 6 labile hydrogens undergo exchange with CH3CH2OD and C6H5CH2OD). Differential labeling of peptides also was accomplished, and the extent of H/D exchange is dependent on the amino acids which are present as well as their order in the chain.     

The shape of leucine in the gas phase.

Two structures of neutral leucine are detected in the jet-cooled rotational spectrum of a laser-ablation molecular-beam Fourier transform microwave (LA-MB-FTMW) experiment. The comparison between the experimental rotational and (14)N nuclear quadrupole coupling constants and those calculated ab initio provides conclusive evidence for the identification of the conformers. The most stable species is stabilized by a N-H...O=C intramolecular hydrogen bond and a cis-COOH interaction, while a higher-energy conformer exhibits a N...H-O intramolecular hydrogen bond and trans-COOH, as in lower aliphatic amino acids. The isobutyl side chain adopts the same configuration in the two conformers of leucine, characterized by a trans arrangement of the C'-C(alpha)-C(beta)-C(gamma)-C(delta) chain. The differences with the preferred side chain configurations observed in valine and isoleucine are discussed.     

Vibrational signature of charge solvation vs salt bridge isomers of sodiated amino acids in the gas phase

The vibrational spectra of the gaseous sodium complexes of glycine (Gly-Na(+)) and proline (Pro-Na(+)) have been recorded in the spectral range 1150-2000 cm(-1). The complexes were formed by matrix-assisted laser desorption-ionization, introduced in the cell of a Fourier transform ion cyclotron resonance mass spectrometer, and their infrared spectra were recorded using photons of variable energy emitted by a free electron laser. Photon absorption was probed by the diminished intensity of the parent ion, due to its infrared-induced dissociation into bare sodium cation and the free amino acid and the appearance of Na(+). The observed absorption bands are assigned using ab initio computations of the IR spectra of the lowest energy isomers in each case. They provide the first experimental evidence that the salt bridge isomer is formed in the case of Pro-Na(+). In contrast, charge solvation by chelation of Na(+) between nitrogen and the carbonyl oxygen seems to be most favorable for Gly-Na(+), but a mixture of isomers cannot be ruled out in this case.     

Infrared fingerprint spectroscopy and theoretical studies of potassium ion tagged amino acids and peptides in the gas phase

Infrared multiple-photon dissociation spectroscopy is effected on the K(+) tagged aromatic amino acids tyrosine and phenylalanine, as well as the K(+) tagged peptides bradykinin fragment 1-5 and [Leu]-enkephalin. The fingerprint (800-1800 cm(-1)) infrared spectra of these species are compared to density-functional theory (DFT) calculated spectra to determine whether the complex is in the charge solvation (CS) or salt bridge (SB) (i.e. zwitterionic) configuration. For the aromatic amino acids the CS structure is favored and the tridentate N/O/ring structure is found to be the preferred binding geometry for K(+). The experimental and theoretical evidence for bradykinin fragment 1-5 tagged with K(+) suggests that the SB structure is favored; the calculations indicate a head-to-tail looped structure stabilized by a salt bridge between the protonated guanidine group and the deprotonated C-terminus, which allows K(+) to sit in a binding pocket with five C=O electrostatic interactions. For K(+) tagged [Leu]-enkephalin the spectroscopic evidence is not as clear. While the calculations clearly favor a CS structure and the observation of a weak carboxylic acid C=O stretching band in the infrared spectrum matches this finding, the prominence of a band at 1600 cm(-1) renders the analysis more ambiguous, and hence the presence of some salt bridge ions cannot be excluded. Another striking feature in the [Leu]-enkephalin spectrum is the high infrared activity of the tyrosine side-chain modes, which can be clearly identified from comparison to the [Tyr + K](+) experimental spectrum, but which is not reproduced by the DFT calculations.     

Determination of amino acids in urine by gas chromatography

This report describes a convenient means of reducing the complex matrix which is responsible for interference during gas chromatographic determination of amino acids in urine. The pre-chromatographic clean-up employs the principle of solid phase extraction using bonded silica incorporating cation exchange groups. This approach avoids the detrimental effects on amino acid recoveries associated with resin-based cation exchangers. In spite of significant reduction in the complexity of chromatograms, only the high efficiency and resolving power offered by the analytical capillary column (e. g. fused silica open tubular, FSOT) is sufficient for quantitative and analysis of amino acids in urine. Reproducibility data from the complete procedure are determined, coefficients of variation (CV) for most amino acids being better than 5% with a mean recovery of 96%.     

Tryptophan analysis simultaneously with other amino acids in gas phase hydrochloric acid hydrolyzates using the Pico-TagTM Work Station

Summary Classical liquid phase hydrolysis of proteins with hydrochloric acid in the presence of tryptamine [3-(2-aminoethyl)indole] has shown that tryptophan can be protected from destruction. An exhaustive study has been made to establish the optimum conditions for protein hydrolysis, in the gas phase using the Pico-Tag^TM Work Station, as a function of time and temperature. The amino acid content, including tryptophan and cyst(e)ine, of standard proteins such as lysozyme, human albumin and -chymotrypsin were determined as phenylthiocarbamyl (PTC) derivatives. On 5 m Hypersil columns (15×4.6 mm) the quantitation of twenty PTC amino acids requires 22 minutes. The reproducibility of the measurements was 5.8% (relative standard deviation) or less.