Molecular mass spectrometric identification of superoxide dismutase in the liver of mice <Emphasis Type="Italic">Mus musculus</Emphasis> and <Emphasis Type="Italic">Mus spretus</Emphasis> using a metallomics analytical approach

This paper reports the identification and quantification of superoxide dismutase in the liver of Mus musculus and Mus spretus mice using a metallomics analytical approach. The approach consisted of using orthogonal chromatographic systems coupled to ICP–MS and UV detectors. Size-exclusion fractionation of the cytosolic extracts was followed by anion-exchange chromatographic separation of Cu- and Zn-containing species. After purification then tryptic digestion, Cu- and Zn-containing superoxide dismutase was identified by nESI-QqTOF. The MS–MS spectra of doubly charged peptides, with the Mascot searching engine, were used to obtain the sequence of the protein.     

The extent and effects of peptide sequence scrambling via formation of macrocyclic <Emphasis Type="Italic">b</Emphasis> ions in model proteins

The extent and effects of sequence scrambling in peptide ions during tandem mass spectrometry (MS/MS) have been examined using tryptic peptides from model proteins. Sequencescrambled b ions appeared in about 35% of 43 tryptic peptides examined under MS/MS conditions. In general, these ions had relatively low abundances with averages of 8% and 16%, depending on the instrumentation used. A few tryptic peptides gave abundant scrambled b ions in MS/MS. However, peptide and protein identifications under proteomic conditions with Mascot were not affected, even for these peptides wherein scrambling was prominent. From the 43 tryptic peptides that have been investigated, the conclusion is that sequence scrambling is unlikely to impact negatively on the accuracy of automated peptide and protein identifications in proteomics.     

Discrimination between wild-type and ampicillin-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis> by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was optimized to discriminate between wild-type and ampicillin-resistant Escherichia coli. Only ampicillin-resistant E. coli displayed an m/z ≈ 29,000 peak, which was confirmed as β-lactamase by in-gel digestion followed by peptide mass fingerprinting. Rapid MALDI-TOF MS detection of antibiotic-resistance could fulfill an important clinical need, providing critical phenotypic information beyond genus–species identification.     

Synthesis of <Emphasis Type="Italic">o</Emphasis>(<Emphasis Type="Italic">m</Emphasis>)-carborane-containing azomethines

A method of preparative synthesis of o(m)-carborane-containing azomethines via the condensation of o(m)-carboranyl-C-methylene-4-formylbenzoates with aliphatic, cycloaliphatic, and aromatic amines was developed.     

Liquid chromatography/mass spectrometry characterization of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Shigella</Emphasis> species

Liquid chromatography/quadrupole time of flight mass spectrometry (LC/QTOF MS) utilizing electrospray ionization was employed to monitor protein expression in Escherichia coli and Shigella organisms. Comparison with MALDI/TOF-MS revealed more proteins, particularly above 15 kDa. A combination of automated charge state deconvolution, spectral mirroring, and spectral subtraction was used to reveal subtle differences in the LC/MS data. Reproducible intact protein biomarker candidates were discovered based on their unique mass, retention time, and relative intensity. These marker candidates were implemented to differentiate closely related strain types, (e.g., two distinct isolates of E. coli O157:H7) and to correctly identify unknown pathogens. This LC/MS approach is less labor-intensive than pulsed-field gel electrophoresis, affords greater specificity than real-time PCR, and requires no primers or antibodies. Additionally, this approach would be beneficial during outbreaks of foodborne disease or bioterrorism investigations by complementing methods typically used in diagnostic microbiology laboratories.     

Excess enthalpies of binary mixtures of <Emphasis Type="Italic">ortho</Emphasis>-, <Emphasis Type="Italic">meta</Emphasis>-, <Emphasis Type="Italic">para</Emphasis>-structural isomers containing aliphatic group

To obtain further systematic information for the isomer systems, the excess molar enthalpies for binary (o + m), (o + p), (m + p)-isomers of methoxymethylbenzene, ethylmethylbenzene, diethylbenzene, chloromethylbenzene, tolunitrile and fluorobenzonitrile, tolylacetonitrile were measured at 298.15 K. In this article, the results are discussed and compared with those of previous works. The excess enthalpies of binary systems in different solid and liquid states were measured when the pure component of o-/m-tolunitrile, fluorobenzonitrile was titrated into the prior (o + p) or (m + p) mixtures. A series calculation for the interaction energies (IE) between the isomers was carried out for the pair molecules by ab initio MO of Gaussian 09. Correlations between the excess enthalpies at a molar fraction of x = 0.5 and the intermolecular energy are discussed.     

Mass-spectra and molecular structure of <Emphasis Type="Italic">O</Emphasis>-alkyl <Emphasis Type="Italic">N,N</Emphasis>-dialkylamidocyanophosphates

The electron impact mass spectra were recorded and the key directions of fragmentation of the highly toxic O-alkyl N,N-dialkylamidocyanophosphates were identified. The sets of characteristic ions for the 10 subgroups of the main group of this class were determined. Based on the values of the mass numbers and peak intensities of characteristic ions generalized spectral images for each of the 10 studied subgroups of this class were simulated. A new method of group identification of O-alkyl N,N-dialkylamidocyanophosphates was developed.     

Synthesis of <Emphasis Type="Italic">m</Emphasis>-carborane-C-carboxylic acid hexahydrobenzo[<Emphasis Type="Italic">a</Emphasis>]acridine esters

The possibility was demonstrated of selective preparation of m-carborane-C-carboxylic acid hexahydrobenzo[a]acridine esters both by the reaction of 1,3-diketones with azomethines obtained by condensation of m-carborane-C-carboxylic acid vanillin and vanillal esters with 1- or 2-naphthylamine and by the cascade heterocyclization of 1- or 2-naphthylamine, m-carborane-C-carboxylic acid vanillin and vanillal esters, and CH-acids.     

Growth Inhibition and Induction of Stress Protein,GroEL, of <Emphasis Type="Italic">Bacillus cereus</Emphasis> Exposed to Antibacterial Peptide Isolated from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> SC-8

This study was conducted to investigate the antibacterial effect of BSAP-254 on Bacillus cereus with the induced stress proteins. The BSAP-254 is an antimicrobial peptide isolated from soybean-fermenting bacteria, Bacillus subtilis SC-8. It had a narrow spectrum of activity against B. cereus group. The growth inhibitory effect of BSAP-254 (50 μg/mL) reduced the population of B. cereus from >10⁸ to 10⁴ colony-forming units per milliliter within 30 min. In B. cereus exposed to BSAP-254, 14 intracellular proteins were differentially expressed as determined by 2-DE coupled with MS. Of the differentially expressed proteins identified, the stress protein GroEL, which is heat shock protein, was induced in B. cereus exposed to antibacterial peptide.     

New lignans from <Emphasis Type="Italic">Syringa reticulata</Emphasis> var. <Emphasis Type="Italic">mandshurica</Emphasis>

In the search for platelet-activating-factor (PAF) antagonists, two new lignan compounds were isolated from the leaves of Syringa reticulata Hara var. mandshurica. Their structures were elucidated as (7R,8S, 8'S)-3,4,3',4'-dimethylenedioxy-8,9-dihydroxy-8.8', 7-O-9'-lignan (mandshuricol A) and (7R,8S,8'S)-3',4'methylenedioxy-4-methoxy-3,8,9-trihydroxy-8.8', 7-O-9'-lignan (mandshuricol B), Mandshuricol A and B showed antagonistic activity on PAF in the [³H] PAF receptor binding assay with IC₅₀ values of 4.8 × 10^–5 M and 3.5 × 10^–5 M, respectively.     

Thermal characterization of starch-based polymers produced by <Emphasis Type="Italic">Ophiostoma</Emphasis> spp

The thermal behavior of modified starches (MS) produced by biosynthetic pathway is described based on a comparative analysis with native starches (NS). MS were produced by fermentation in presence of Ophiostoma spp. cultures. Thermogravimetric analysis (TG) with successive derivatives (DTG) and differential scanning calorimetry (DSC) were used for this study. NS results showed a single peak dominating both the TG (DTG) and DSC plots. A double thermal transition event was detected in samples of MS. The procedural decomposition temperature (T _i–T _f; lowest onset temperature of initial and final mass change) was carried out within a narrow interval of temperatures for NS (610–640 °C). This interval could not be reached within the 1,000 °C range in MS. Residues higher than 10% were recorded for MS at this temperature. The presence of the double thermal transition in MS is discussed.     

<Emphasis Type="SmallCaps">d</Emphasis>-Amino Acids in Animal Peptides

Summary. Secreted peptides from diverse sources have been found to contain a d-amino acid. From the sequence of cloned mRNAs coding for the precursors of such peptides it could be deduced that in all cases tested so far the d-amino acid in the final product is derived from the corresponding l-amino acid present in the primary product of translation. Enzymes catalyzing such an l- to d-isomerization in peptide linkage have been isolated from the venom of a spider and the skin secretions of frogs. Even though these are completely different proteins, the reaction mechanism is the same, namely a de-protonation/re-protonation of the α-carbon of an amino acid with concomitant inversion of the chirality. Sequences potentially coding for homologues of the frog enzyme are present in the genome of different vertebrate species.     

<Emphasis Type="Italic">Z</Emphasis>-and <Emphasis Type="Italic">E</Emphasis>-conformers of <Emphasis Type="Italic">N</Emphasis>-chloroacetylcytisine

N-Chloroacetylcytisine was synthesized by acylation of (–)-cytisine. Stable Z- and E-conformers with respect to rotational isomerism around the N-12–CO bond were found in PMR spectra at room temperature. The point at which PMR resonances of the Z- and E-conformers coalesced upon heating was measured. The transition barrier between the conformers was estimated.     

Backbone and side-chain specific dissociations of <Emphasis Type="Italic">z</Emphasis> ions from non-tryptic peptides

Backbone z-type fragment ions formed by electron-transfer dissociation (ETD) of doubly protonated peptides AAHAL, AHDAL, and AHADL were subjected to collisional activation and their dissociation products were studied by ETD-CID-MS³ and MS⁴. Electron structure theory calculations were performed to elucidate ion structures and reaction mechanisms. All z ions showed competitive eliminations of C₃H₇ and C₄H₈ from the C-terminal Leu side chain. The energetics and kinetics of these dissociations were studied computationally for the z₄ ion from AAHAL, and optimized structures are reported for several intermediates and transition states. RRKM calculations on the combined B3LYP and PMP2/6-311++G(2d,p) potential energy surface provided unimolecular rate constants that closely reproduced the experimental branching ratios for C₃H₇ and C₄H₈ eliminations. Mechanisms were also studied for the loss of CO₂ from z ions generated by ETD of AHDAL and AHADL and for a specific radical-induced Asp-C_α-CO backbone cleavage. CID of the z ions under study did not produce any fragment ions that would indicate cascade backbone dissociations triggered by the radical sites. In contrast, the majority of backbone dissociations occurred at bonds that were remote from the radical sites (spin-remote dissociations) and were triggered by proton migrations that were analogous to those considered for standard peptide ion fragmentations.     

Thermochemical study on ternary complex of dysprosium <Emphasis Type="Italic">m</Emphasis>-nitrobenzoic acid with <Emphasis Type="Italic">o</Emphasis>-phenanthroline

A ternary binuclear complex of dysprosium chloride hexahydrate with m-nitrobenzoic acid and 1,10-phenanthroline, [Dy(m-NBA)₃phen]₂·4H₂O (m-NBA: m-nitrobenzoate; phen: 1,10-phenanthroline) was synthesized. The dissolution enthalpies of [2phen·H₂O(s)], [6m-HNBA(s)], [2DyCl₃·6H₂O(s)], and [Dy(m-NBA)₃phen]₂·4H₂O(s) in the calorimetric solvent (V_DMSO:V_MeOH = 3:2) were determined by the solution–reaction isoperibol calorimeter at 298.15 K to be \Updelta_\texts H_\textm^q \Updelta_{\text{s}} H_{\text{m}}^{\theta } [2phen·H₂O(s), 298.15 K] = 21.7367 ± 0.3150 kJ·mol⁻¹, \Updelta_\texts H_\textm^q \Updelta_{\text{s}} H_{\text{m}}^{\theta } [6m-HNBA(s), 298.15 K] = 15.3635 ± 0.2235 kJ·mol⁻¹, \Updelta_\texts H_\textm^q \Updelta_{\text{s}} H_{\text{m}}^{\theta } [2DyCl₃·6H₂O(s), 298.15 K] = −203.5331 ± 0.2200 kJ·mol⁻¹, and \Updelta_\texts H_\textm^q \Updelta_{\text{s}} H_{\text{m}}^{\theta } [[Dy(m-NBA)₃phen]₂·4H₂O(s), 298.15 K] = 53.5965 ± 0.2367 kJ·mol⁻¹, respectively. The enthalpy change of the reaction was determined to be \Updelta_\textr H_\textm^q = 3 6 9. 4 9 ±0. 5 6   \textkJ·\textmol ^{- 1} . \Updelta_{\text{r}} H_{\text{m}}^{\theta } = 3 6 9. 4 9 \pm 0. 5 6 \;{\text{kJ}}\cdot {\text{mol}}^{ - 1} . According to the above results and the relevant data in the literature, through Hess’ law, the standard molar enthalpy of formation of [Dy(m-NBA)₃phen]₂·4H₂O(s) was estimated to be \Updelta_\textf H_\textm^q \Updelta_{\text{f}} H_{\text{m}}^{\theta } [[Dy(m-NBA)₃phen]₂·4H₂O(s), 298.15 K] = −5525 ± 6 kJ·mol⁻¹.     

Molecular properties of the copolymers of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-diallyl-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylammonium chloride and maleic acid

The hydrodynamic and conformational properties of molecules of poly(N,N-diallyl-N,N-dimethylammonium chloride) and N,N-diallyl-N,N-dimethylammonium chloride-maleic acid copolymers of different compositions in solutions with various ionic-strength and pH values, as well as of the polyelectrolyte complex based on the copolymer with dodecyl sulfate anions in chloroform, are studied. For poly(N,N-diallyl-N,N-dimethylammonium chloride) molecules in a 1 M NaCl solution, the Kuhn segment length and the hydrodynamic diameter of the chain are estimated as A = 3.9 nm and d = 0.48 nm, respectively. In acidic solutions with pH 3.5, the copolymers demonstrate behavior typical for polyelectrolytes. In an alkaline solution with pH 13, when 1 M NaCl is added to the solution of the copolymer containing 29 mol % maleic acid units, there is an antipolyelectrolyte effect that manifests itself as an increase in the intrinsic viscosity of the copolymer and in the hydrodynamic radius of its molecules. It is found that an increase in the fraction of maleic acid units in the copolymer from 12 to 42 mol % brings about a reduction in the equilibrium rigidity of its macromolecules from 4.1 to 2.2 nm. The equilibrium rigidity of polyelectrolyte-complex molecules is higher than that of initial copolymer molecules owing to steric interactions arising between the aliphatic chains of dodecyl sulfate anions. In an electric field, the molecules of the complex are oriented owing to the induced dipole moment resulting from the displacement of dodecyl sulfate anions along the chain contour.     

Substrate Specificity and Thermostability of the Dehairing Alkaline Protease from <Emphasis Type="Italic">Bacillus pumilus</Emphasis>

An alkaline protease (DHAP) from Bacillus pumilus has shown great potential in hide dehairing. To get better insights on its catalytic properties for application, the substrate specificity and thermostability were investigated using five natural proteins and nine synthetic peptides. The results showed that DHAP could hydrolyze five proteins tested here in different specificity. Collagen, a component of animal skin, was more resistant to hydrolysis than casein, fibrin, and gelatin. Among the synthetic peptides, the enzyme showed activity mainly with tetrapeptide substrates with the catalytic efficiency in order of Phe>Leu>Ala at P1 site, although k _m value for AAVA-pN is much lower than that for AAPL-pN and AAPF-pN. With tripeptide substrates, smaller side-chain group (Gly) at P1 site was not hydrolyzed by DHAP. The enzyme showed good thermostability below 60 °C, and lost activity so quickly above 70 °C. The thermostability was largely dependent on metal ion, especially Ca²⁺, although other ions, like Mg²⁺, Mn²⁺, and Co²⁺, could sustain stability at certain extent within limited time. Cu²⁺, Fe²⁺, as well as Al³⁺, did not support the enzyme to retain activity at 60 °C even in 5 min. In addition, the selected metal ions could coordinate calcium in improvement or destruction of thermostability for DHAP.     

Excess enthalpies of binary mixtures of <Emphasis Type="Italic">ortho</Emphasis>-, <Emphasis Type="Italic">meta</Emphasis>-, and <Emphasis Type="Italic">para</Emphasis>-structural isomers

The excess molar enthalpies of 8 binary mixtures for the o-, m-, and p-isomers of fluoroiodobenzene, fluoromethoxybenzene, bromofluorobenzene, chlorofluoro-benzene, difluorobenzene, fluoromethylbenzene, fluoronitrobenzene, and aminofluoro-benzene were measured at 298.15 K. The changes of the measured enthalpies were very small. The experimental results revealed that the isomers containing two electron-acceptor groups showed the most positive excess enthalpy change, while isomers containing both one electron donor and one electron acceptor group, such as aminofluorobenzene, showed more stable and always the most negative results.     

Isolation and characterization of the cellulose synthase genes <Emphasis Type="Italic">PpCesA6</Emphasis> and <Emphasis Type="Italic">PpCesA7</Emphasis> in <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Analysis of cellulose biosynthesis using molecular approaches has been successful in identifying genes in many cellulose-producing organisms, yet the mechanism of cellulose biosynthesis still remains to be understood. We are interested in developing the moss Physcomitrella patens as a useful system for the study of cellulose biosynthesis. This moss affords a number of advantages including a haploid dominated gametophyte and a very high efficiency of homologous recombination in its nuclear DNA for constructing gene knockouts. In addition, P. patens has only a primary cell wall unlike Arabidopsis thaliana, which has both a primary and a secondary cell wall. We identified two full-length cellulose synthase (CesA) genes of P. patens, PpCesA6 and PpCesA7 from an EST database and have analyzed the genomic sequences. PpCesA6 and PpCesA7 show high similarity to each other, both at the cDNA and genomic DNA levels. Single and double knockouts of PpCesA6 and PpCesA7 were generated and screened for phenotypic changes. While the PpCesA6 and PpCesA7 single knockouts did not show any obvious phenotypic differences from the wild-type, the double knockout had significantly reduced stem length. These results suggest that PpCesA6 and PpCesA7 probably have a very similar role in cellulose biosynthesis and their functions may be redundant. Additionally, their roles may overlap with the other P. patens CesAs as observed for CesAs involved in primary cell wall biosynthesis in A. thaliana.     

Electron-Transfer Secondary Reaction Matrices for MALDI MS Analysis of <Emphasis Type="Italic">Bacteriochlorophyll a</Emphasis> in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> and Its Zinc and Copper Analogue Pigments

Bacteriochlorophyll a (BChl a), a photosynthetic pigment performing the same functions of chlorophylls in plants, features a bacteriochlorin macrocycle ring (18 π electrons) with two reduced pyrrole rings along with a hydrophobic terpenoid side chain (i.e., the phytol residue). Chlorophylls analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is not so straightforward since pheophytinization (i.e., release of the central metal ion) and cleavage of the phytol–ester linkage are invariably observed by employing protonating matrices such as 2,5-dihydroxybenzoic acid, sinapinic acid, and α-cyano-4-hydroxycinnamic acid. Using BChl a from Rhodobacter sphaeroides R26 strain as a model system, different electron-transfer (ET) secondary reaction matrices, leading to the formation of almost stable radical ions in both positive ([M]^+?) and negative ([M]^??) ionization modes at m/z 910.55, were evaluated. Compared with ET matrices such as trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), 2,2':5',2''-terthiophene (TER), anthracene (ANT), and 9,10-diphenylanthracene (DP-ANT), 1,5-diaminonaphthalene (DAN) was found to provide the highest ionization yield with a negligible fragmentation. DAN also displayed excellent ionization properties for two metal ion-substituted bacteriochlorophylls, (i.e., Zn- and Cu-BChl a at m/z 950.49 and 949.49), respectively. MALDI MS/MS of both radical charged molecular species provide complementary information, thus making analyte identification more straightforward.

Open image in new window

Graphical Abstract ?