A mechanistic investigation of the enhanced cleavage at histidine in the gas-phase dissociation of protonated peptides

Enhanced gas-phase cleavage of peptides adjacent to histidine was investigated. The peptides examined were angiotensins III (RVYIHPF) and IV (VYIHPF) as well as synthetic peptide analogues with altered key residues ((R)VYI-X-Z-F; X = F or H and Z = A, P, or Sar) or a fixed charge M3P(+)CH(2)C(O)-VYIHPF. While all singly protonated peptide ions containing both histidine and arginine fragment nonselectively, the doubly protonated peptide ions with arginine and histidine, and the singly protonated peptides containing histidine but not arginine, cleave in a selective manner. In particular, dominant complementary b+/y+ product ions resulting from cleavage between the HP amide bond are observed. For the fixed-charge derivative, selective cleavage occurs only if a proton is added to produce a doubly charged precursor. The results are consistent with involvement of a protonated histidine in the selective cleavage. The ratio of b+/y+ is determined by the identity of the residue C-terminal to histidine and by the ability of protonated histidine to transfer a proton to the C-terminal leaving fragment. This was probed further by systematically changing the residue C-terminal to histidine and by alkylating histidine. The results indicate that while b+/y+ complementary ion pairs dominate in doubly protonated RVYIHPF, b5(2+) and b6(2+) product ions dominate the spectra of doubly protonated RVYIHAF. Also, dominant b5(2+) product ions are observed when the histidine side chain is alkylated (H) in doubly protonated RVYIHPF. Based on all of the results, a selective fragmentation mechanism for enhanced cleavage at histidine involving an atypical b ion structure is proposed.     

Differentiating N-terminal aspartic and isoaspartic acid residues in peptides

Formation of isoaspartic acid (isoAsp) is a common modification of aspartic acid (Asp) or asparagine (Asn) residue in proteins. Differentiation of isoAsp and Asp residues is a challenging task owing to their similar properties and identical molecular mass. It was recently shown that they can be differentiated using ion-electron or ion-ion interaction fragmentation methods (ExD) because these methods provide diagnostic fragments c + 57 and z(?) - 57 specific to the isoAsp residue. To date, however, the presence of such fragments has not been explored on peptides with an N-terminal isoAsp residue. To address this question, several N-terminal isoAsp-containing peptides were analyzed using ExD methods alone or combined with chromatography. A diagnostic fragment [M + 2H - 74](+?) was observed for the doubly charged precursor ions with N-terminal isoAsp residues. For some peptides, identification of the N-terminal isoAsp residue was challenging because of the low diagnostic ion peak intensity and the presence of interfering peaks. Supplemental activation was used to improve diagnostic ion detection. Further, N-terminal acetylation was offered as a means to overcome the interference problem by shifting the diagnostic fragment peak to [M + 2H - 116](+?).     

Localization of the O-glycosylated sites in peptides by fixed-charge derivatization with a phosphonium group

The present study demonstrates that matrix-assisted laser desorption ionization/postsource decay (MALDI/PSD) analysis of the molecular cation of glycopeptides derivatized at their amino terminus with a phosphonium group cleaves peptide backbone without removing the glycan. The predictable a-type fragment ions retain the glycan moiety, enabling unambiguous localization of O-glycans on the peptide chain. In contrast, collision-activated dissociation tandem mass spectrometry analysis carried out on the doubly charged protonated phosphonium cation results in the predominant loss of the sugar moiety from the peptide. This result supports the previously proposed charge-induced fragmentation mechanism of the sugar-peptide bond. MALDI/PSD analysis of glycopeptides converted to their acetyl phosphonium derivatives is an effective alternative to electron capture dissociation, as illustrated by the positioning of up to three GalNac residues along the full tandem repeat peptide sequence derived from the MUC 5AC mucin.     

Stochastic energy transition of peptide bond under the action of hydrolytic enzyme

Stochastic energy transition of peptide bond (PB) in enzyme-substrate complex (ESC) is investigated. By introducing the Pippard model and thermal fluctuation as the noise, the two important mechanisms of enhancing PB breaking rate, i.e., the Fermi resonance and the reduction of potential barrier height due to oxygen ion O⁻ are studied. The necessary frequency ratio 1:2 for Fermi resonance is verified by using the deterministic averaging method. With the noise terms present, the average energy transition rate of PB is predicted by using the stochastic averaging method combined with solving the Pontryagin equation or assuming the PB breaking as a Poisson process. Comparison of the numerical results with those from Monte Carlo simulations shows that the theoretical analysis is effective. To include the influence of the oxygen ion on the PB breaking, a proposal is presented to describe the vibration of the oxygen ion and to couple it with PB vibration. In terms of the coupled system, the two mechanisms may be investigated in a unified framework.     

Benzoyl derivatization as a method to improve retention of hydrophilic peptides in tryptic peptide mapping

This study exploits the increase in chromatographic retention that accrues from benzoyl derivatization of primary amines as a tool to increase sequence coverage in tryptic peptide mapping. N-hydroxysuccinamide sulfonyl benzoate quantitatively derivatizes primary amines of peptides. Introduction of the hydrophobic benzoyl moiety into peptides increased retention of peptides during reversed-phase chromatography (RPC), particularly in the case of smaller hydrophilic peptides. Short chain (1-6 amino acids) tryptic fragments of model proteins lysozyme, myoglobin, and cytochrome c derivatized with N-hydroxysuccinamide sulfonyl benzoate eluted in the linear acetonitrile gradient. Application of benzoyl derivatization was further extended to achieve complete sequence coverage of a therapeutic protein, recombinant human growth hormone, and in detection of single amino acid polymorphism.     

Use of performic acid oxidation to expand the mass distribution of tryptic peptides

Significant identification of proteins by mass fingerprinting and partial sequencing of tryptic peptides is central to proteomics. However, peptide masses cluster with distances of approximately 1 Da. Expanding these clusters will give more peptides of unique masses, thereby identifying proteins with a higher significance. The mass clusters can be expanded downward by including more oxygen atoms in the peptides. Classic performic acid oxidation modifies three residues, Cys to CysO(3), Met to MetO(2), and Trp to TrpO(2). In this study, we compare the mass distributions of tryptic peptides computed from the predicted proteomes of Bacillus subtilis, Drosophila melanogaster, Arabidopsis thaliana, and Homo sapiens modified by oxidation, reduction, and reduction followed by carboxymethylation, carboxamidomethylation, or pyridylethylation. Forty to 46% of the eukaryotic tryptic peptides contain Cys, Met, or Trp. Additionally, the importance of mass accuracy of differentially modified tryptic peptides for significant protein identification by database searches was analyzed. The results show that performic acid oxidation gives markedly extended mass distributions at mass accuracies from +/-0.002 to +/-0.25 Da for the eukaryotes. The effect of the expanded mass distribution on significant protein identification was illustrated by searching simulated mass peak lists against the databases containing oxidized and reduced tryptic peptides. The specificity of formic acid oxidation was tested experimentally, and no general adverse effects were detected. Tryptic peptides provided a 100% sequence coverage of oxidized barley grain peroxidase by LC-MS, and the sequence coverages of oxidized and carboxymethylated bovine serum albumin were similar by MALDI-TOF MS analyses.     

In situ surface-enhanced infrared study of hydrogen bond pairing of complementary nucleic acid bases at the electrochemical interface

Surface-enhanced infrared absorption spectroscopy with a Kretschmann-type attenuated total reflection configuration has been used to study hydrogen-bonded pairing between 6-amino-8-purinethiol, a thiol-derivatized adenine, immobilized on a gold electrode surface, and thymidine, a complimentary base derivative of adenine, in 0.1 M NaClO4 aqueous solution as a function of applied potential. 6-Amino-8-purinethiol is adsorbed on a gold surface via a sulfur atom to form a S-Au bond. Nearly half of the adsorbed molecules are protonated, and the long axis of the adenine moiety is tilted from the surface normal at open circuit potential. As the potential increases, the acid-base equilibrium is shifted toward the unprotonated form and the adenine moiety is reoriented toward a nearly perpendicular configuration. The hydrogen bond interaction between the adsorbed 6-amino-8-purinethiol with thymidine in solution is greatly affected by the protonation and orientation of the adenine moiety and is controllable by the applied potential. Due to steric hindrance, an adenine-thymine-type hydrogen bond pair is formed only at potentials more positive than 0.1 V (vs SCE) where the unprotonated adenine moiety is perpendicularly oriented.     

Elution-modified displacement chromatography coupled with electrospray ionization-MS: on-line detection of trace peptides at low-femtomole level in peptide digests

Elution-modified displacement chromatography (EMDC) was employed to achieve peptide separations with high efficiency. On-line ESI-MS and ESI-MS/MS measurements showed enrichment and detection of kemptide, a protein kinase A peptide substrate, at low femtomole levels when it was added as a trace marker component to a tryptic digest of bovine serum proteins or to a human growth hormone peptide digest at concentration ratios between 1:10(5) and 1:10(6). In another EMDC separation, five peptides were detected in a mixture containing 20 fmol of human growth hormone tryptic digest mixed with the bovine serum protein digest. We found that EMDC facilitated rapid detection and sequence analysis of trace peptides at levels of approximately 0.5 fmol/microL in complex peptide mixtures with a wide dynamic concentration range. Accordingly, the detection of kemptide by EMDC was found to be 3-4 orders of magnitude more sensitive than that attained in conventional linear elution chromatography separations performed with the same peptide loads. Kemptide was phosphorylated in vitro and was detected along with its neutral loss product in peptide mixtures at low femtomole levels. EMDC enabled both detection and amino acid sequence determination on trace levels of phosphorylated and other posttranslationally modified peptides, suggesting that the technique may be useful for proteomics applications where detection and analysis of trace level peptides are problematic.     

A thermochemical approach to the gas-phase reduction of metal sulphates

The reduction of different metal sulphates by hydrogen occurs at different temperatures and can lead to a variety of products. It has been shown that the starting temperatures for the reduction of several sulphates are directly related to the magnitude of their (exothermic) heats of formation per equivalent. The thermochemical data also provide a basis for the interpretation of the relative thermal stabilities (under nitrogen atmosphere) of several metal sulphates. A consideration of the appropriate heat of formation values can also be useful in understanding the different reduction products obtained for different metal sulphates. Finally, the reduction behaviour of some binary metal sulphates is also consistent with the thermochemical data.     

Self-assembled nanoparticles for cellular delivery of peptide nucleic acid using amphiphilic N,N,N-trimethyl-O-alkyl chitosan derivatives

Peptide nucleic acid (PNA) holds enormous potentials as antisense/antigenic drug due to its specific binding ability and biostability with DNA or RNA. However, the poor cellular delivery is the key obstacle in development of PNA therapy. To overcome this difficulty, we developed self-assembled nanoparticles (NPs) for delivery of PNA to living cells using amphiphilic CS derivatives. A series of N,N,N-trimethyl-O-alkyl chitosans (TMACs) with different lengths of alkyl chains were synthesized. The structures of these synthesized chemicals were characterized with FT-IR and ¹H NMR. We found that the TMACs were all able to self-assemble in aqueous condition to form nano-size NPs. These nano-size NPs are spherical shape with a size range of around 100?nm and a zeta potential above +30?mV. PNA was easily encapsulated into chitosan derivative NPs by an ultrasonic method with entrapment efficiency up to 75%. The PNA-loaded TMAC NPs released the drug in a sustained manner in PBS (pH 7.4) at 37?°C. N,N,N-trimethyl-O-cetyl chitosan (TMCC) showed the best in vitro hemocompatibility and cell viability. These TMCC based NPs were able to dramatically increase the cellular uptake of PNA, specifically, 66-fold higher compared to without using these nanoparticles. The results suggest that the designed TMCC NPs might be a promising solution for improving cellular delivery of PNA.

Open image in new window

     

An examination of the linkage of cleavage cracks at grain boundaries

Abstract

Grain boundaries resist the propagation of cleavage cracks in polycrystalline materials, and 3D geometrical models have been used to predict the accommodation required at a grain boundary as a crack propagates from grain to grain. This paper describes how focused ion beam (FIB) microscopy, which provides topographic and crystallographic contrast imaging and allows ion milling to be undertaken at selected areas of interest, can be used to investigate these local fracture events. Results of low temperature fracture of polycrystalline bcc Fe–3%Si and hcp zinc are presented. The interactions between these results and the geometrical modelling are briefly discussed.     

Analysis of glycosyl bond cleavage and related isotope effects in collision-induced dissociation quadrupole-time-of-flight mass spectrometry of isomeric trehaloses

The configuration isomers alpha,alpha-, alpha,beta-, and beta,beta-trehalose are distinguishable by a relative ion abundance analysis using collision-induced dissociation MS/MS measurements in electrospray ionization quadrupole-time-of-flight mass spectrometry. The relative abundance of the Y-type fragment ion of alpha,alpha-trehalose is the highest and that of beta,beta-trehalose is the lowest, indicating that alpha-glycosyl bonds cleave more easily than beta-glycosyl bonds. The relative ion abundance depends on both the alpha- and beta-glycosyl linkage type and the number of alpha-glycosyl bonds. The reaction path of glycosyl bond cleavage is calculated computationally using the molecular orbital method in the form of Hartree-Fock theory in conjunction with the 6-31G(d) basis set. The results are consistent with the experimental data. Isotope effects on the fragmentation of the glycosyl bonds are detected in the experiments of the H2O/D2O solvent systems. Furthermore, the isotope effect regarding beta,beta-trehalose is larger than those of alpha,alpha- and alpha,beta-trehalose, indicating that the isotope effect on the beta-glycosyl bond cleavage is larger than that on the alpha-glycosyl bond cleavage. The thermal energy increase in trehalose-d8 molecules over the corresponding trehalose molecules is calculated from the vibrational modes.     

Incorporation of salicylic acid derivatives to hydrophilic copolymer systems with biomedical applications

Hydrogels based on polymeric derivatives of salicylic acid have been prepared for biomedical applications by free radical copolymerization of 2-hydroxy-4-methacrylamidobenzoic acid, 4HMA, and 2-hydroxy-5-methacrylamidobenzoic acid, 5HMA, with 2- hydroxyethylmethacrylate, HEMA, in a wide range of compositions. The reactivity ratios of 4HMA and 5HMA with HEMA in radical copolymerization processes have been determined from their ¹H NMR spectra by applying linearization methods and non-linear least square treatments. Tgs of the corresponding copolymers were analyzed by DSC. The swelling behavior in water of the prepared copolymers was studied in comparison to poly-(HEMA), poly-(4HMA) and poly-(5HMA) hydration degrees, being in all cases superior to 35%. The hydrolytical behavior of the synthesized copolymers was studied at three different pHs (2, 7.4 and 10) determining the release percentage of the salicylic acid derivatives, 4-amino salicylic acid, 4ASA, and 5-amino salicylic acid, 5ASA, analyzed by high performance liquid chromatography (HPLC). The release analysis was followed during 230 days and a pH dependence was observed obtaining the highest release percentages at pH=10, whereas at physiological pH (7.4) the release percentages were in range from 2 to 5% at that time for all copolymer systems. The hydrolytical stability is enough for long-term applications like bone cements, ionomers, etc. © 2001 Kluwer Academic Publishers     

Enhancement of MALDI-MS spectra of C-terminal peptides by the modification of proteins via an active ester generated in situ from an oxazolone

For selective C-terminal derivatization of peptides and proteins, we have devised a method for activating the C-terminal carboxyl group by extending the oxazolone chemistry. A mixture of formic acid and acetic anhydride was found to be effective for the formation of an oxazolone, which was converted to an active ester in situ in the presence of a phenol or an N-hydroxide. In particular, the resulting active ester with pentafluorophenol facilitated the subsequent reaction with an amine and the hydrazine derivative to yield the C-terminal amide and hydrazide, respectively. The peptides thus coupled with arginine methyl ester or 2-hydrazino-2-imidazoline containing the guanidino moiety exhibited the positive-ion peaks in matrix-assisted laser desorption/ionization (MALDI) mass spectra with appreciably enhanced intensities. As expected from the reaction mechanism, the carboxyl groups of aspartic and glutamic acid residues were not modified, while the amino groups that could react with the activated peptides were concomitantly protected by formylation. The MALDI peaks corresponding to the C-terminal peptide fragments of proteins were specifically enhanced, discriminating against those from internal peptides that were not tagged with a positive charge. In favorable cases, the C-terminal peptide fragments were clearly discerned by MALDI-MS after chymotryptic digestion and were identified by their MALDI postsource decay analysis. Based on these results, we suggest a method for C-terminal sequencing of a protein.     

Ion mobility spectrometry of gas-phase ions from laser ablation of solids in air at ambient pressure

A mobility spectrometer was used to characterize gas-phase ions produced from laser ablation of solids in air at 100 degrees C and at ambient pressure with a beam focused to a diameter of 相似文献   

Template-directed synthesis of mesoporous silicas containing phosphonic acid derivatives in the surface layer

A template-directed process, using 1-dodecylamine as a template, is developed for the synthesis of mesoporous silicas containing the phosphonic acid derivatives ≡Si(CH₂)₂P(O)(OC₂H₅)₂ and ≡Si(CH₂)₃P(O)(CH₃)(ONa) in the surface layer. The porous materials obtained by removing the template with boiling methanol have specific surfaces of 854 and 505 m²/g, accessible pore volumes of 0.42 and 0.37 cm³/g, and pore diameters of 2.2 and 2.5 nm, respectively. As shown by scanning electron microscopy and x-ray diffraction, the mesoporous silicas are nonuniform in particle shape and size, and their structure is less ordered than that of classic mesoporous silicas, such as MCM-41. IR and ¹³C, ³¹P, and ²⁹Si CP/MAS NMR spectroscopy data indicate that, in the surface layer of the mesoporous silica prepared with the use of ≡Si(CH₂)₂P(O)(OC₂H₅)₂, the functional groups are present in the form of T ² and T ³ structural units. In addition, the surface layer contains alkoxy groups and water, which participates in hydrogen bonding.     

Characterization of the acid/base and redox chemistry of phytochelatin analogue peptides

The phytochelatins are a family of polydisperse, thiol-rich peptides that are synthesized by plants in response to exposure to heavy metals. The amino acid sequence of the phytochelatin peptides is (gamma-glutamyl-cysteinyl)n-glycine, where n typically ranges from 2 to 5. In the first phase of a program to characterize the coordination chemistry of the phytochelatins with heavy metals, the phytochelatin analogue peptides acetyl(gamma-glutamyl-cysteinyl)n-glycine amide (Ac-(gamma-Glu-Cys)n-NH2, n = 2-6) have been synthesized by solid-phase peptide synthesis methods and characterized by 1H NMR spectroscopy. The 1H NMR spectra of the analogue peptides were completely assigned by using band-selective homonuclear-decoupled (BASHD) two-dimensional NMR experiments to achieve spectral resolution. The acid dissociation constant of each cysteine residue in each peptide was determined from chemical shift-pH titration data for the CalphaH protons of the cysteine residues. The resonances for the CalphaH protons were resolved in BASHD-total correlation spectroscopy spectra that were measured as a function of pH. The pKA values for a given thiol group depend on the position of the cysteine residue in the sequence, with the thiol group of the cysteine residue attached to the C-terminal glycine being the most acidic. The pKA values also depend on the size of the peptide, increasing as the size, and thus the negative charge, of the peptide increases. The redox potential for oxidation of the two thiol groups of Ac(gamma-Glu-Cys)2-NH2 to form an intramolecular disulfide bond was also determined by measuring the equilibrium constant for its thiol/disulfide exchange reaction with glutathione.     

Biosynthesis of polyamides containing amino acid residues through the specific aminolysis of amino acid ester derivatives

The enzymatic polycondensation of polyamides containing amino acid residues was demonstrated. The diamide monomers, containing amino acid (Phe, Leu, Val) esters at both ends, were synthesized using a Scotten–Baumann reaction. Polymerization was conducted through an enzymatic aminolysis of the ester bond by aliphatic diamine in organic solvents. Several proteases were employed to evaluate the enzyme specificity on the polymerization. α-Chymotrypsin was found to have the most effective catalyst activity on the monomer containing phenylalanine, reflecting the specific enzymatic process. Approximate molecular weight at about 7000 was obtained through this enzymatic polycondensation. The structures of the obtained polymers were confirmed employing IR and NMR spectra. The polymerization occurred in various solvents, including two- and one-phase systems. It is found that water content in organic solvent affects the yield.     

Use of a designed peptide array to infer dissociation trends for nontryptic peptides in quadrupole ion trap and quadrupole time-of-flight mass spectrometry

Observed peptide gas-phase fragmentation patterns are a complex function of many variables. To systematically probe this phenomenon, an array of 40 peptides was synthesized for study. The array of sequences was designed to hold certain variables (peptide length) constant and randomize or balance others (peptide amino acid distribution and position). A high-quality tandem mass spectrometry (MS/MS) data set was acquired for each peptide for all observed charge states on multiple MS instruments, quadrupole-time-of-flight and quadrupole ion trap. The data were analyzed as a function of total charge state and number of mobile protons. Previously known dissociation trends were observed, validating our approach. In addition, the general influence of basic amino acids on dissociation could be determined because, in contrast to the more widely studied tryptic peptides, the amino acids H, K, and R were positionally distributed. Interestingly, our results suggest that cleavage at all basic amino acids is suppressed when a mobile proton is available. Cleavage at H becomes favored only under conditions where a partially mobile proton is present, a caveat to the previously reported trend of enhanced cleavage at H. Finally, all acquired data were used as a benchmark to determine how well these sequences would have been identified in a database search using a common algorithm, Mascot.