A matrix-assisted laser desorption/ionization compatible reagent for tagging tryptophan residues

Chemical tagging of amino acids is an important tool in proteomics analysis, and has been used to introduce isotope labels and mass defect labels into proteolytic peptides by derivatization of cysteine or lysine residues. Here, we present a new reagent with chemical specificity for tryptophan residues. Previously, 2-nitrobenzenesulfenyl chloride has been used as a highly specific reagent for labeling tryptophan residues. We show that this tag undergoes UV dissociation during matrix assisted laser desorption/ionization (MALDI). The multiplicity of photofragments increases the difficulty of characterizing the derivatization products. To overcome this problem, we have synthesized a new reagent, 2-(trifluoromethyl)benzenesulfenyl chloride, which is shown to react quantitatively with tryptophan in peptides and proteins. Most significantly, it exhibits high photostability in MALDI-Fourier transform mass spectrometry analyses.     

Impact of environmental conditions on the marine natural product bryostatin 1

Marine Natural Products (MNPs), such as bryostatin 1, are exposed to a range of physical and chemical conditions through the life cycle of the host organism. These include exposure to sunlight, oxidizing and reducing agents, cation binding, and adsorption to reactive metal oxide surfaces. Using Fourier Transform-Ion Cyclotron Resonance (FT-ICR), Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS), UV/Vis absorbance spectroscopy, and molecular modeling, we studied the impact of UV light, TiO2, I2, and reaction with FeCl3 on the structure of bryostatin 1. Our results demonstrate that natural conditions transform bryostatin to a number of structures, including one with a molar mass of 806 Da, which we have previously identified in the sediment collected from the Gulf of Mexico. To date, at least 20 different structures of bryostatin have been reported in the literature. This work suggests that these variations may be products of the chemical environment in which the bryozoa Bugula neritina resides and are not the result of genetic variations within Bugula.     

Quality-control materials in the USDA National Food and Nutrient Analysis Program (NFNAP)

The US Department of Agriculture (USDA) Nutrient Data Laboratory (NDL) develops and maintains the USDA National Nutrient Databank System (NDBS). Data are released from the NDBS for scientific and public use through the USDA National Nutrient Database for Standard Reference (SR) (). In 1997 the NDL initiated the National Food and Nutrient Analysis Program (NFNAP) to update and expand its food-composition data. The program included: 1) nationwide probability-based sampling of foods; 2) central processing and archiving of food samples; 3) analysis of food components at commercial, government, and university laboratories; 4) incorporation of new analytical data into the NDBS; and 5) dissemination of these data to the scientific community. A key feature and strength of the NFNAP was a rigorous quality-control program that enabled independent verification of the accuracy and precision of analytical results. Custom-made food-control composites and/or commercially available certified reference materials were sent to the laboratories, blinded, with the samples. Data for these materials were essential to ongoing monitoring of analytical work, to identify and resolve suspected analytical problems, to ensure the accuracy and precision of results for the NFNAP food samples.     

Tuning from pi,pi to charge-transfer excited states in styryl-substituted terthiophenes: an ultrafast and steady-state emission study

The steady-state and transient emission properties of unsubstituted terthiophene and a series of 3'-[E-2-(4-R-phenyl)ethenyl]-2,2':5',2' '-terthiophenes (where R = H, MeO, NH(2), CN, NMe(2), NO(2)) have been examined. The R = NO(2) compound is nonemissive at room temperature in all solvents but cyclohexane. All of the other compounds show measurable steady-state emission in a variety of solvents. The behavior of these spectra may be split into two groups. The first group, those substituted compounds with R = CN, NH(2) and NMe(2), show solvatochromic behavior, where their Lippert-Mataga plots suggest changes in dipole upon photoexcitation ranging from 12.5 to 16.0 D. For the second group, where R = H and MeO (and unsubstituted terthiophene as well), the Lippert-Mataga plots indicate dipole moment changes ranging from 0 to 7.9 D. The difference in behavior between the two groups of emissive compounds can be attributed to a charge-transfer character of the emitting state in the first group. This conclusion is supported by density functional theory calculations, which show that the frontier MOs in the group one compounds are spatially separated whereas those of group two have frontier MOs that are delocalized over both the styryl and terthiophene moieties. Picosecond time-resolved fluorescence spectroscopy reveals that unsubstituted terthiophene has the shortest emission lifetime of 140 ps in acetonitrile. For the styryl substituted terthiophenes, the lifetimes are much longer and range from 320 to 670 ps for R = CN and NMe(2) respectively, a result that can be explained in terms of a smaller rate of intersystem crossing in these compounds.     

A Novel Screening Method for Competitive FRET-Aptamers Applied to <Emphasis Type="Italic">E. coli</Emphasis> Assay Development

A novel high-throughput screening method is described in which a family of DNA aptamers selected against E. coli outer membrane proteins (OMPs) is subjected to PCR in the presence of fluorophore-dUTP conjugates using Deep Vent® exo- polymerase. The fluorophore-doped aptamers and their complementary strands are then heated to render them single-stranded and screened in filter well microtiter plates for fluorescence resonance energy transfer (FRET) assay potential. Using this system, a superior competitive FRET-aptamer designated EcO 4R was identified and the location of its putative binding pocket was determined by individually testing FRET potential in each of the secondary loop structures. By labeling the binding pocket with Alexa Fluor (AF) 647 and binding the aptamer to heavily Black Hole Quencher-3 (BHQ-3)-labeled E. coli bacteria, detection of as few as 30 live unlabeled E. coli per ml was achieved in a competitive displacement FRET assay format. The far red fluorescence emission enables detection in largely blue-green autofluorescent matrices. In addition, the competitive transfer of AF 647-EcO-4R aptamer to unlabeled E. coli cells after a 15 min equilibration period was verified by fluorescence microscopy. The present study also demonstrated that high aptamer affinity is not well correlated with competitive FRET potential.     

Minimal bending energies of bilayer polyhedra

Motivated by recent experiments on bilayer polyhedra composed of amphiphilic molecules, we study the elastic bending energies of bilayer vesicles forming polyhedral shapes. Allowing for segregation of excess amphiphiles along the ridges of polyhedra, we find that bilayer polyhedra can indeed have lower bending energies than spherical bilayer vesicles. However, our analysis also implies that, contrary to what has been suggested on the basis of experiments, the snub dodecahedron, rather than the icosahedron, generally represents the energetically favorable shape of bilayer polyhedra.     

The electronic structures of commensurate Ru(0001)–(3 × 3)–4Kr and Ru(0001)–(5 × 5)–Kr using density functional theory

We have calculated the minimum energies for each of three positionings of the adatom unit cells for Ru(0001)–(3 × 3)–4Kr (high Kr coverage) and for Ru(0001)–(5 × 5)–Kr (low Kr coverage). The differences between the results for the low and high-coverage cases may clarify puzzles posed by the experimental results of Narloch and Menzel. The low-coverage solution converges to a structure having Kr in the top site at a height of 3.09 Å above the substrate with the adsorption energy 185 meV. In the high-coverage case, adatom unit cells with a corner Kr at top, fcc hollow, and hcp hollow locations are found to have nearly the same adsorption energy of 175 meV. The height of the corner atom above the substrate is found to be 3.35, 3.54, and 3.50 Å for the top, fcc hollow and hcp hollow sites, respectively. These results are explained by demonstrating that there is an enhancement of the substrate electronic density of states at krypton orbital energies in the low-coverage case.     

Einen Apparat zur schnellen Bestimmung der Viscosit?t von Schmier?len

Ohne Zusammenfassung     

Gas-phase photoacoustic sensor at 8.41 μm using quartz tuning forks and amplitude-modulated quantum cascade lasers

We demonstrate the performance of a novel infrared photoacoustic laser absorbance sensor for gas-phase species using an amplitude-modulated quantum cascade (QC) laser and a quartz tuning fork microphone. The photoacoustic signal was generated by focusing 5.3 mW of a Fabry–Pérot QC laser operating at 8.41 μm between the tines of a quartz tuning fork which served as a transducer for the transient acoustic pressure wave. The sensitivity of this sensor was calibrated using the infrared absorber Freon 134a by performing a simultaneous absorption measurement using a 31-cm absorption cell. The power and bandwidth normalized noise equivalent absorption sensitivity (NEAS) of this sensor was determined to be D=2.0×10^-8 W cm^-1/Hz^1/2. A corresponding theoretical analysis of the instrument sensitivity is presented and is capable of quantitatively reproducing the experimental NEAS, indicating that the fundamental sensitivity of this technique is limited by the noise floor of the tuning fork itself. PACS 43.60.Vx; 43.58.Wc; 43.58.Hp; 84.40.Xb