Dissecting the proline effect: dissociations of proline radicals formed by electron transfer to protonated Pro-Gly and Gly-Pro dipeptides in the gas phase

We report a combined experimental and computational study of the proline effect in model dipeptides Pro-Gly and Gly-Pro. Gas-phase protonated peptide ions were discharged by glancing collisions with potassium or cesium atoms at 3 keV collision energies, and the peptide radical intermediates and their dissociation products were analyzed following collisional ionization to anions. The charge reversal (+CR-) mass spectra of (Pro-Gly + H)+(1a+) and (Gly-Pro + H)+ (2a+) showed dramatic differences and thus provided a sensitive probe of ion structure. Whereas 1a+ completely dissociated upon charge inversion, 2a+ gave a nondissociated anion as the most abundant product. Ab initio and density functional theory calculations provided structures and vertical recombination energies (REvert) for 1a+ and 2a+. The recombination energies, REvert = 3.07 and 3.36 eV for 1a+ and 2a+, respectively, were lower than the alkali metal ionization energies and indicated that the collisional electron transfer to the peptide ions was endoergic. Radical 1a* was found to exist in a very shallow local energy minimum, with transition state energies for loss and migration of H indicating very facile dissociation. In contrast, radical 2a* was calculated to spontaneously isomerize upon electron capture to a stable dihydroxycarbinyl isomer (2e*) that can undergo consecutive and competitive isomerizations by proline ring opening and intramolecular hydrogen atom transfers to yield stable radical isomers. Radical 2e* and its stable isomers were calculated to have substantial electron affinities and thus can form the stable anions that were observed in the +CR- mass spectra. The calculated TS energies and RRKM kinetic analysis indicated that peptide N-C alpha bond dissociations compete with pyrrolidine ring openings triggered by radical sites at both the N-terminal and C-terminal sides of the proline residue. Open-ring intermediates were found in which loss of an H atom was energetically preferred over backbone dissociations. This provided an explanation for the proline effect causing low incidence of electron capture dissociations of N-C alpha bonds adjacent to proline residues in tryptic peptides and also for some peculiar behavior of proline-containing protein cation-radicals.     

Electron super-rich radicals in the gas phase. A neutralization-reionization mass spectrometric and ab initio/RRKM study of diaminohydroxymethyl and triaminomethyl radicals

Diaminohydroxymethyl (1) and triaminomethyl (2) radicals were generated by femtosecond collisional electron transfer to their corresponding cations (1+ and 2+, respectively) and characterized by neutralization-reionization mass spectrometry and ab initio/RRKM calculations at correlated levels of theory up to CCSD(T)/aug-cc-pVTZ. Ion 1+ was generated by gas-phase protonation of urea which was predicted to occur preferentially at the carbonyl oxygen with the 298 K proton affinity that was calculated as PA = 875 kJ mol-1. Upon formation, radical 1 gains vibrational excitation through Franck-Condon effects and rapidly dissociates by loss of a hydrogen atom, so that no survivor ions are observed after reionization. Two conformers of 1, syn-1 and anti-1, were found computationally as local energy minima that interconverted rapidly by inversion at one of the amine groups with a <7 kJ mol-1 barrier. The lowest energy dissociation of radical 1 was loss of the hydroxyl hydrogen atom from anti-1 with ETS = 65 kJ mol-1. The other dissociation pathways of 1 were a hydroxyl hydrogen migration to an amine group followed by dissociation to H2N-C=O* and NH3. Ion 2+ was generated by protonation of gas-phase guanidine with a PA = 985 kJ mol-1. Electron transfer to 2+ was accompanied by large Franck-Condon effects that caused complete dissociation of radical 2 by loss of an H atom on the experimental time scale of 4 mus. Radicals 1 and 2 were calculated to have extremely low ionization energies, 4.75 and 4.29 eV, respectively, which belong to the lowest among organic molecules and bracket the ionization energy of atomic potassium (4.34 eV). The stabilities of amino group containing methyl radicals, *CH2NH2, *CH(NH2)2, and 2, were calculated from isodesmic hydrogen atom exchange with methane. The pi-donating NH2 groups were found to increase the stability of the substituted methyl radicals, but the stabilities did not correlate with the radical ionization energies.     

Diorganoruthenium complexes incorporating noninnocent [C(6)H(2)(CH(2)ER(2))(2)-3,5](2)(2-) (E = N, P) bis-pincer bridging ligands: synthesis, spectroelectrochemistry, and DFT studies

The dinuclear complex [(tpy)RuII(PCP-PCP)RuII(tpy)]Cl2 (bridging PCP-PCP = 3,3',5,5'-tetrakis(diphenylphosphinomethyl)biphenyl, [C6H2(CH2PPh2)2-3,5]22-) was prepared via a transcyclometalation reaction of the bis-pincer ligand [PC(H)P-PC(H)P] and the Ru(II) precursor [Ru(NCN)(tpy)]Cl (NCN = [C6H3(CH2NMe2)2-2,6]-) followed by a reaction with 2,2':6',2' '-terpyridine (tpy). Electrochemical and spectroscopic properties of [(tpy)RuII(PCP-PCP)RuII(tpy)]Cl2 are compared with those of the closely related [(tpy)RuII(NCN-NCN)RuII(tpy)](PF6)2 (NCN-NCN = [C6H2(CH2NMe2)2-3,5]22-) obtained by two-electron reduction of [(tpy)RuIII(NCN-NCN)RuIII(tpy)](PF6)4. The molecular structure of the latter complex has been determined by single-crystal X-ray structure determination. One-electron reduction of [(tpy)RuIII(NCN-NCN)RuIII(tpy)](PF6)4 and one-electron oxidation of [(tpy)RuII(PCP-PCP)RuII(tpy)]Cl2 yielded the mixed-valence species [(tpy)RuIII(NCN-NCN)RuII(tpy)]3+ and [(tpy)RuIII(PCP-PCP)RuII(tpy)]3+, respectively. The comproportionation equilibrium constants Kc (900 and 748 for [(tpy)RuIII(NCN-NCN)RuIII(tpy)]4+ and [(tpy)RuII(PCP-PCP)RuII(tpy)]2+, respectively) determined from cyclic voltammetric data reveal comparable stability of the [RuIII-RuII] state of both complexes. Spectroelectrochemical measurements and near-infrared (NIR) spectroscopy were employed to further characterize the different redox states with special focus on the mixed-valence species and their NIR bands. Analysis of these bands in the framework of Hush theory indicates that the mixed-valence complexes [(tpy)RuIII(PCP-PCP)RuII(tpy)]3+ and [(tpy)RuIII(NCN-NCN)RuII(tpy)]3+ belong to strongly coupled borderline Class II/Class III and intrinsically coupled Class III systems, respectively. Preliminary DFT calculations suggest that extensive delocalization of the spin density over the metal centers and the bridging ligand exists. TD-DFT calculations then suggested a substantial MLCT character of the NIR electronic transitions. The results obtained in this study point to a decreased metal-metal electronic interaction accommodated by the double-cyclometalated bis-pincer bridge when strong sigma-donor NMe2 groups are replaced by weak sigma-donor, pi-acceptor PPh2 groups.     

Capillary electrophoresis mass spectrometry coupling with immobilized enzyme electrospray capillaries

Open tubular capillary enzyme reactors were studied for rapid protein digestion and possible on-line integration into a CE/ESI/MS system. The need to minimize the time of the analyte molecules to diffuse towards the surface immobilized enzyme and to maximize the surface-to-volume (S/V) ratio of the open tubular reactors dictated the use of very narrow bore capillaries. Extremely small protein amounts (atto-femtomoles loaded) could be digested with enzymes immobilized directly on the inside wall of a 10 microm I.D. capillary. Covalently immobilized L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-trypsin and pepsin A were tested for the surface immobilization. The enzymatic activity was characterized in the flow-through mode with on-line coupling to electrospray ionization-time of flight-mass spectrometer (ESI/TOF-MS) under a range of protein concentrations, buffer pH's, temperatures and reaction times. The optimized reactors were tested as the nanospray needles for fast identification of proteins using CE-ESI/TOF-MS.     

Monolithic Stationary Phases for Capillary Electrochromatography Based on Synthetic Polymers: Designs and Applications

Monolithic materials have quickly become a well‐established stationary phase format in the field of capillary electrochromatography (CEC). Both the simplicity of their in situ preparation method and the large variety of readily available chemistries make the monolithic separation media an attractive alternative to capillary columns packed with particulate materials. This review summarizes the contributions of numerous groups working in this rapidly growing area, with a focus on monolithic capillary columns prepared from synthetic polymers. Various approaches employed for the preparation of the monoliths are detailed, and where available, the material properties of the resulting monolithic capillary columns are shown. Their chromatographic performance is demonstrated by numerous separations of different analyte mixtures in variety of modes. Although detailed studies of the effect of polymer properties on the analytical performance of monolithic capillaries remain scarce at this early stage of their development, this review also discusses some important relationships such as the effect of pore size on the separation performance in more detail.     

Morphology,crystal structure,and orientation of poly-p-oxybenzoate on mica

Polymerization of p-acetoxybenzoic acid between mica sheets, from both the melt and dilute solution, in a wide range of polymerization temperatures (180–400°C) yields an epitaxial-like overgrowth of PpOBA on the mica cleavage surface. The PpOBA overgrowth forms long rows by lateral aggregation of ribbon crystals situated in three substrate directions rotated by 60°. The c-axes of the PpOBA phase I and II lattices are parallel to the 001 plane of the mica, coinciding with the a-axes of the mica hexagonal structure. The a_I- and b_II-axes lie predominantly on the mica surface. The surface-orienting effect is tentatively attributed to surface steps and or/polar effects; lattice matching does not occur. The recrystallization of bulk polymer samples previously prepared also leads to ordering between mica like that in melt or solution polymerization. © 1997 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 35: 1807–1820, 1997     

Comparative study of ZnO layers prepared by PLD from different targets at various oxygen pressure levels

Two series of polycrystalline zinc oxide (ZnO) layers, from Zn or ZnO targets, were grown on silicon (1 1 1) substrates by pulsed laser deposition (PLD) at ambient oxygen pressure levels, stepwise increased from 1 to 35 Pa. For ablation of targets, a pulsed Nd:YAG laser was used. The structural and morphological properties of the layers were investigated by scanning electron microscopy (SEM), atomic force microscopy (AFM), and secondary ion mass spectrometry (SIMS). The SEM images of ZnO layers in SE mode show a uniform granular structure and modified surface morphology, depending on oxygen pressure. The mean grain size in height and lateral directions decreases with an increase of oxygen pressure from 1 to 5 Pa, while a subsequent rise of oxygen pressure from 5 to 35 Pa will cause an increase in the grain size. The AFM measurement revealed that the surface structures of zinc oxide layers grown from different targets were similar, and the layers formed at an ambient oxygen pressure of 5 Pa exhibited the smallest values of calculated roughness and granularity. SIMS depth profiling analyses confirmed that the ZnO composition was homogenous across the layer, up to the abrupt change of chemical composition at the interface between the ZnO layer and the Si substrate.      

Femtosecond multicolor transient absorption spectroscopy of colloidal silver nanoparticles

Time-resolved femtosecond multicolor absorption spectroscopy of silver nanoparticle (NP) colloids with particle diameter in range of 10–30 nm is presented. The amplified femtosecond excitation of the surface plasmon resonance band resulted in transient absorption spectra reflecting the electron-phonon relaxation dynamics, which takes place on the early picosecond time scale. The monitored band with enhanced absorption in the 490–540 nm spectral range exhibited red-shift with increasing pump fluency from 0.4 mJ/cm² to the 1.5 mJ/cm² level. The growth of the relaxation time with increasing pump fluency reveals the temperature dependent relaxation dynamics caused by the nanometer sized electron confinement in the case of silver. This effect was confirmed also by identification of the relaxation time dependence on the particle diameter at constant pump fluency. The complex experimental results revealed nonlinearities both in the laser excitation and electron relaxation processes.     

Application of MEKC and monolithic CEC for the analysis of bioactive naphthoquinones in Eleutherine americana

Two microscale separation techniques for the analysis of bioactive naphthoquinones in Eleutherine americana were developed and validated. By MEKC four compounds (eleuthoside B, isoeleutherin, eleutherol and eleutherinoside A) could be determined in plant extracts using an aqueous electrolyte solution composed of 25 mM sodium tetraborate, 50 mM sodium cholate and 20% THF. CEC on a polymeric methacrylate‐based monolith with strong cationic properties showed promising results, as it additionally enabled the separation of two enantiomers, eleutherin and isoeleutherin. The mobile phase for CEC experiments comprised 3 mM ammonium formate in a mixture of ACN and water. At an applied voltage of ?25 kV, all five markers were baseline separated in less than 12 min. Both methods were successfully validated for linearity (MEKC: R²≥0.999; CEC: R²≥0.997), sensitivity (MEKC: LOD=4–5 μg/mL; CEC: LOD=2–8 μg/mL), accuracy (MEKC: 96.5–102.7% recovery; CEC: 97.1–103.5% recovery) and precision (MEKC: σ_rel≤2.43%; CEC: σ_rel≤2.21%). The quantitative analysis of naphthoquinone derivatives in several E. americana samples showed that both methods are suitable for practical applications, because the results were well comparable to those obtained by established techniques such as HPLC.     

Preparative separation of a multicomponent peptide mixture by mass spectrometry

We report on the first multiplex preparative separation by mass spectrometry of bio-organic molecules in the 200-350 Da mass range that is typical for synthetic drugs. A five-component mixture consisting of two di- and three tripeptides has been separated by mass using a specially designed mass spectrometer. The instrument for preparative separations consists of an electrospray ionization (ESI) source, ion transfer optics, an electrostatic sector, and an inhomogeneous-field magnetic mass analyzer that achieves linear mass dispersion of ion beams. Protonated peptides produced by electrospray were separated, nondestructively landed on a 16-channel array of dry collector plates, and reconstituted in solution. The preparation procedures and the instrumental conditions have been optimized to maximize the ion currents. The significant features of the special mass spectrometer are high ion currents and simultaneous separation and collection of mixture components.