Optimization of a MALDI TOF-TOF mass spectrometer for intact protein analysis

A MALDI TOF-TOF instrument was optimized and evaluated for intact protein analysis by tandem mass spectrometry. Ion source voltages and delay times were adjusted to affect an up to a 10-fold improvement in fragment ion yield compared to data obtained using default settings employed in peptide analysis. For large peptides (3-4.5 kDa), up to 90% of all possible b- and y-fragment ions were observed, which provides sufficient information for de novo sequencing and unambiguous protein identification. Product ion signals associated with preferential cleavages C-terminal to aspartic acid and glutamic acid residues and N-terminal to proline residues became dominant with increased protein molecular weight. Matrix effects were also evaluated and, among the eight matrices examined, alpha-cyano-4-hydroxycinnamic acid (CHCA) was found to produce the best intact protein tandem mass spectra for proteins up to 12 kDa. Optimized performance yielded detection limits of 50-125 fmol for proteins of 4 and 12 kDa, respectively. This improved performance has yielded an instrument with potential to be a useful tool in proteomic investigations via analysis of intact proteins.     

Speciation analysis of tellurium by solid-phase extraction in the presence of ammonium pyrrolidine dithiocarbamate and inductively coupled plasma mass spectrometry

Under acidic conditions tellurium(IV) formed a complex with ammonium pyrrolidine dithiocarbamate (APDC). The tellurium(IV) complex was completely retained on a non-polar Isolute silica-based octadecyl (C(18)) sorbent-containing solid-phase extraction (SPE) cartridge, while the uncomplexed Te(VI) passed through the cartridge and remained as a free species in the solution. Only partial Te(IV) was retained on the SPE cartridge for samples without addition of APDC. On the basis of different retention behaviours of the complexed Te(IV) and uncomplexed Te(VI), a simple and highly sensitive method is proposed for the determination of total tellurium and Te(VI) by SPE separation and inductively coupled plasma mass spectrometry (ICP-MS) detection. The Te(IV) concentration was calculated as the difference between total tellurium and Te(VI) concentrations. The detection limit (3 sigma) is 3 ng L(-1) tellurium. Factors affecting the separation and detection of tellurium species were investigated. Coexisting ions did not show significant interferences with the Te(IV)-APDC complex retention and the subsequent ICP-MS detection of Te. The method has been successfully applied to the tellurium speciation analysis in waters with spiked recoveries for Te(IV) and Te(VI) of 86.0-108% and 87.1-97.4%, respectively.     

New polythiophenes bearing electron-acceptor phthalocyanine chromophores

Two new donor-acceptor copolymers comprising a polythiophene backbone, and bearing phthalocyanine chromophores on the side chains have been prepared. Preliminary photophysical characterization of these materials by FTIR photoinduced absorption indicates that electron transfer from the polythiophene to the phthalocyanine units takes place.     

Complete-active-space self-consistent-field/Amber parameterization of the Lys296–retinal–Glu113 rhodopsin chromophore-counterion system

A special hybrid quantum mechanics/molecular mechanics forcefield is defined, parameterized and validated for studying the photoisomerization path of the retinal chromophore in the rhodopsin protein. It couples a multireference ab initio Hamiltonian (CASSCF and second-order multireference many-body perturbation theory using a CASSCF reference) to describe the chromophore while the rest of the protein is approximated with the Amber forcefield. The frontier has been carefully parameterized in order to reproduce full quantum mechanics torsional energy profiles, for both the ground state and the first excited state. It is also shown that replacing the chromophore counterion with point charges is a valid approximation. This result is interpreted in terms of a cancellation effect for which a possible explanation is given.     

Characterization of Carbohydrates in Paper and Paper Pulps Using Anion Exchange Chromatography and Principal Component Analysis

In the course of this investigation, a method for the characterization and differentiation of paper pulps was developed. After hydrolyzation of the papers and the respective raw pulps with trifluoroacetic acid, the hydrolyzates were analyzed with respect to their carbohydrate composition using ion exchange chromatography. The variations in carbohydrate composition mainly arise from the various hemicelluloses of the papers and pulps. The chromatographic results were then further processed using principal component analysis which allows correlating the various papers to different pulp materials.     

The Mitsunobu Inversion Reaction of Sterically Hindered 17-Hydroxy Steroids

The Mitsunobu inversion reaction of 3-methoxyestra-1,3,5(10)-trien-17-ol is dramatically influenced by the acidic component. There appears to be a relationship between the dissociation constant of the electron-withdrawing substituent on the aryl acid and the overall effectiveness of the reaction, with more acidic species generally providing a higher yield of inverted product.     

Characterization of a Nudix hydrolase from <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> with a marked specificity for (deoxy)ribonucleoside 5'-diphosphates

Background  

Nudix hydrolases form a protein family whose function is to hydrolyse intracellular nucleotides and so regulate their levels and eliminate potentially toxic derivatives. The genome of the radioresistant bacterium Deinococcus radiodurans encodes 25 nudix hydrolases, an unexpectedly large number. These may contribute to radioresistance by removing mutagenic oxidised and otherwise damaged nucleotides. Characterisation of these hydrolases is necessary to understand the reason for their presence. Here, we report the cloning and characterisation of the DR0975 gene product, a nudix hydrolase that appears to be unique to this organism.     

Physico-chemical characterization of liposomes with covalently attached hepatitis A VP3(101-121) synthetic peptide.

The covalent conjugation of a 20-mer peptide belonging to the VP3 capsid protein of hepatitis A virus to the surface of preformed liposomes was investigated. Three different bonds (disulfide, thioether and amide) were established between the peptide sequence and liposomes bearing at their surface appropriate reactive groups. The effect of the relative concentration of the N-[4-(p-maleimidophenyl)butyryl]dipalmitoylphosphatidylethanolamine anchor in liposomes on stability during coupling of the peptide sequence was studied. The interaction of the three liposomal preparations with phospholipids in a biomembrane model system, monolayers at the air-water interface, is also reported. The results showed that although the peptides associate with liposomes in similar yields for the three strategies studied, differences can be observed when their interaction with phospholipid monolayers composed of dipalmitoylphosphatidylcholine is analysed.     

Resistance distance and Laplacian spectrum

The resistance distance r _ij between two vertices v _i and v _j of a (connected, molecular) graph G is equal to the resistance between the respective two points of an electrical network, constructed so as to correspond to G, such that the resistance of any two adjacent points is unity. We show how the matrix elements r _ij can be expressed in terms of the Laplacian eigenvalues and eigenvectors of G. In addition, we determine certain properties of the resistance matrix R=||r _ij ||. AcknowledgementsThis research was supported by the Natural Science Foundation of China and Fujian Province, and by the Ministry of Sciences, Technologies and Development of Serbia, within Project no. 1389. The authors thank Douglas J. Klein (Galveston) for useful comments.     

Photocatalytic TiO2 Coatings: Effect of Substrate and Template

Transparent TiO₂ films with a high photodegradation activity towards an azo dye in aqueous solution were prepared by sol–gel processing. Films on soda–lime glass supports protected with a thin silica barrier layer exhibited better crystallization and monodisperse nanoparticles, higher absorption of light below 370 nm, and higher photocatalytic activity than those films deposited on bare glass supports proving the detrimental effect of interdiffused sodium ions on the development of the anatase nanostructure. The effect of substrate was more pronounced in thinner films (300 nm) than in thicker ones (1200 nm), which were achieved by adding a template (i.e. Pluronic F127) to the sol.