Use of two-dimensional liquid fractionation for separation of proteins from cell lysates without the presence of methionine oxidation

A novel two-dimensional (2D) chromatographic method is developed to separate proteins from malignant breast cancer whole cell lysates. Protein mixtures are first separated according to their pIby chromatofocusing followed by an orthogonal non-porous reversed-phase separation. An important advantage of this 2D chromatographic method is that, unlike gel-based methods, it does not result in methionine oxidation. The lack of methionine oxidation during separation is demonstrated by the analysis of protein tryptic digests using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS. Our novel 2D chromatographic method used in combination with on-target light-induced methionine oxidation provides a means for studying methionine-containing peptides. Methionine residues in peptide sequences are partially oxidized with light exposure. Neither the location nor the modification of methionine in the peptide sequence affects the oxidation. As a result, multiple peaks are observed in MALDI-TOF-MS spectra after light exposure. Sequence information derived from light-induced methionine can be applied to enhance the database search results obtained through peptide mass fingerprinting.     

Integral equation theory for two-dimensional polymer melts

The polymer reference interaction site model theory is investigated for two-dimensional polymer melts composed of freely-jointed hard disk chains and tangent-disk rods. Exact results for the intramolecular pair correlation functions are input into the theory, and predictions of the theory for the intermolecular pair correlation functions are tested via comparison with simulation. The theory is not as accurate for this system as it is for three-dimensional polymer melts, and the quantitative predictions are not good except at the highest area fractions. Possible reasons for the deficiency in the theory are discussed.     

Two-stage dilute-acid pretreatment of softwoods

Whole treechips obtained from softwood forest thinnings were pretreated via single-and two-stage dilute-sulfuric acid pretreatment. Whole-tree chips were impregnated with dilute sulfuric acid and steam treated in a 4-L steam explosion reactor. In single-stage pretreatment, wood chips were treated using a wide range of severity. In two-stage pretreatment, the first stage was carried out at low severity tomaximize hemicellulose recovery. Solubilized sugars were recovered from the first-stage prehydrolysate by washing with water. In the second stage, water-insoluble solids from first-stage prehydrolysate were impregnated with dilute sulfuric acid, then steam treated at more severe conditions to hydrolyze a portion of the remaining cellulose to glucose and to improve the enzyme digestibility. The total sugar yields obtained after enzymatic hydrolysis of two-stage dilute acid-pretreated samples were compared with sugar yields from single-stage pretreatment. The overall sugar yield from two-stage dilute-acid pretreatment was approx 10% higher, and the net enzyme requirement was reduced by about 50%. Simultaneous saccharification and fermentation using an adapted Saccharomyces cerevisiae yeast strain further improved cellulose conversion yield and lowered the enzyme requirement.     

Extraction and characterization of adenovirus

A new methodology for the extraction and characterization of proteins from Coomassie-stained sodium dodecylsulfate polyacrylamide gel electrophoresis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been described. The utility of this methodology was demonstrated in the characterization of adenovirus proteins. The key steps in the extraction and destaining process involve washing the excised band with a combination of solvents that include 10% acetic acid, acetonitrile, methanol, and formic acid:water:isopropanol mixture. By using this procedure, we determined adenovirus proteins with molecular weights ranging from 10,000 to 110,000 Da by MALDI-MS, obtaining a detection limit of approximately 6 pmol. Parallel experiments were successfully carried out to analyze adenovirus proteins from Cu-stained gels. It was observed that increase in laser intensity resulted in significant improvements in the quality of MALDI mass spectra for the analysis of inefficiently destained proteins from Cu-stained gels.     

An innovative method for measuring hydrogen and deuterium: Chemical reaction interface mass spectrometry with nitrogen reactant gas

A new method for measuring deuterium isotopic enrichment with CRIMS (chemical reaction interface mass spectrometry) is described. Using nitrogen as the reactant gas in a chemical reaction interface generates molecular hydrogen that provides the H2 and HD from which the deuterium content can be analyzed with a benchtop quadrupole mass spectrometer. Samples of deuterated leucine in unlabeled leucine were used as the primary test species. Detection of deuterium enrichment was accurate, precise, and linear. We used this scheme to evaluate the results of a process to acetylate lysine residues in a peptide-neurotensin. With separation on a C18 column, we found a 61% yield of the desired monoethylated product that had a D/H ratio very close to the theoretical one. Isotope ratio monitoring for deuterated species will be important in metabolism studies where CRIMS generates a comprehensive and quantitative view of products of deuterated precursors. Where concerns about metabolic isotope effects of deuterium are absent, the use of deuterium will enable these studies to be performed with simpler syntheses and at less cost than if using 13C or 15N.     

Parallel Detection of Different DNA Sequences on One Gold Electrode

Motivated by the potential of electrochemical techniques to analyze hybridization events fast and in a simple and cost‐effective way we present here a detection system allowing a parallel electrochemical DNA analysis. For this purpose different probe DNA strands have been immobilized on one electrode. By the use of two different target DNA sequences, both marked with the redox active methylene blue, we can show that hybridization with the complementary probe sh“NA strands can occur without steric hindrance. Each target has been recognized down to 3nM with a very high specificity of the sensor. In addition, we can detect two different ssDNA targets labeled with different redox active molecules, methylene blue and ferrocene, on one sensor surface simultaneously.     

Bonding structure of phenylacetylene on hydrogen-terminated Si(111) and Si(100): surface photoelectron spectroscopy analysis and ab initio calculations

Interfaces between phenylacetylene (PA) monolayers and two silicon surfaces, Si(111) and Si(100), are probed by X-ray photoelectron spectroscopy (XPS), ultraviolet photoelectron spectroscopy (UPS), and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy, and the results are analyzed using ab initio molecular orbital calculations. The monolayer systems are prepared via the surface hydrosilylation reaction between PA and hydrogen-terminated silicon surfaces. The following spectral features are obtained for both of the PA-Si(111) and PA-Si(100) systems: a broad π-π* shakeup peak at 292 eV (XPS), a broad first ionization peak at 3.8 eV (UPS), and a low-energy C 1s → π* resonance peak at 284.3 eV (NEXAFS). These findings are ascribed to a styrene-like π-conjugated molecular structure at the PA-Si interface by comparing the experimental data with theoretical analysis results. A conclusion is drawn that the vinyl group can keep its π-conjugation character on the hydrogen-terminated Si(100) [H:Si(100)] surface composed of the dihydride (SiH(2)) groups as well as on hydrogen-terminated Si(111) having the monohydride (SiH) group. The formation mechanism of the PA-Si(100) interface is investigated within cluster ab initio calculations, and the possible structure of the H:Si(100) surface is discussed based on available data.     

Coupling protein complex analysis to peptide based proteomics

Proteolysis is a central component of most proteomics methods. Unfortunately much of the information relating to the structural diversity of proteins is lost during digestion. This paper describes a method in which the native proteome of yeast was subjected to preliminary fractionation by size exclusion chromatography (SEC) prior to trypsin digestion of SEC fractions and reversed phase chromatography-mass spectral analysis to identify tryptic peptides thus generated. Through this approach proteins associated with other proteins in high molecular mass complexes were recognized and identified. A focus of this work was on the identification of Hub proteins that associate with multiple interaction partners. A critical component of this strategy is to choose methods and conditions that maximize retention of native structure during the various stages of analysis prior to proteolysis, especially during cell lysis. Maximum survival of protein complexes during lysis was obtained with the French press and bead-beater methods of cell disruption at approximately pH 8 with 200 mM NaCl in the lysis buffer. Structure retention was favored by higher ionic strength, suggesting that hydrophobic effects are important in maintaining the structure of protein complexes. Recovery of protein complexes declined substantially with storage at any temperature, but storage at -20°C was best when low temperature storage was necessary. Slightly lower recovery was obtained with storage at -80°C while lowest recovery was achieved at 4°C. It was concluded that initial fractionation of native proteins in cell lysates by SEC prior to RPC-MS/MS of tryptic digests can be used to recognize and identify proteins in complexes along with their interaction partners in known protein complexes.     

Effects of Ultraviolet A-1 Radiation on Calcineurin Activity and Cytokine Production in (Skin) Cell Cultures†

Calcineurin (Cn) is the target of immunosuppressive drugs used for maintenance therapy of transplant patients. UV radiation is also known to be immunosuppressive and, like the Cn inhibitors, UV has been shown to positively influence various inflammatory skin diseases. Recently, Cn activity has been demonstrated in skin and skin cell cultures. In the present study we have investigated the effects of UV(A-1) irradiation on Cn activity in skin. In total skin we found a significant reduction in Cn activity after exposure to 450 kJ m⁻² of UVA-1 (340–400 nm). In repeated experiments cultures of fibroblasts and keratinocytes also showed dose-dependent and selective reduction in Cn activity after UVA-1 irradiation. UVB irradiation caused a decrease in the Cn activity of one of two fibroblast cultures and was ineffective in keratinocytes. In Jurkat cells and PBMC UVA-1 reduced Cn activity and also the production of cytokines such as interleukin (IL)-2, γ-interferon, IL-4 and IL-10 that are controlled by the Ca²⁺–Cn pathway. These results indicate that UV(A-1) irradiation may lead to inactivation of Cn in the skin and thus suppress the skin immune system in a similar fashion to the Cn inhibitors.     

Infinite Polyiodide Chains in the Pyrroloperylene–Iodine Complex: Insights into the Starch–Iodine and Perylene–Iodine Complexes

We report the preparation and X‐ray crystallographic characterization of the first crystalline homoatomic polymer chain, which is part of a semiconducting pyrroloperylene–iodine complex. The crystal structure contains infinite polyiodide I_∞^δ?. Interestingly, the structure of iodine within the insoluble, blue starch–iodine complex has long remained elusive, but has been speculated as having infinite chains of iodine. Close similarities in the low‐wavenumber Raman spectra of the title compound and starch–iodine point to such infinite polyiodide chains in the latter as well.