Identification of selenium in the leaf protein of sunflowers by a combination of 2D-PAGE and laser ablation ICP-MS

We report on a method for the identification of selenium-containing proteins in an extract of sunflower leafs. It is based on the separation of the proteins by 2-dimensional gel electrophoresis, followed by detection of selenium via laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The laser system was operated in a raster mode at 100?μm?s^-1 and proved to be an efficient alternative in the search for selenoproteins in the spots of the gels. The instrumental parameters were optimized in terms of plasma energy and application of optimal reaction cell conditions, and the detection of the mass ⁸⁰Se¹⁶O⁺ which enabled the elimination of interfering species. Selenium was identified in 9.6% of the analyzed spots, indicating its random incorporation into the primary structure of the proteins.

Analysis of narcotic drugs in biological samples using hollow fiber liquid�Cphase microextraction and gas chromatography with nitrogen phosphorus detection

We report on a method for trace analysis of the narcotic drugs alfentanil, fentanyl, and sufentanil in plasma and urine. Two?Cphase hollow fiber liquid?Cphase microextraction was combined with GC using nitrogen?Cphosphorus detection. Experimental parameters were optimized to give a viable analytical procedure whose limits of detection range from 8 to 15?ng L^?1 (at an S/N of 3). The calibration curves are linear between 0.1 and 50???g L^?1, with squared correlation coefficients (r ²) between 0.9953 and 0.9979. Precision values range from 2.4% to 3.3% (intra?Cday RSD) and 3.2 to 6.3% (inter?Cday RSD). The relative recoveries varied from 27.8% to 84.6% (for spiked plasma) and 75 to 85.2% (for spiked urine). The method consumes little solvent, is simple, fast, inexpensive, and well suitable for the analysis of complicated matrices.

Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.

Functionalized manganese-doped zinc sulfide quantum dot-based fluorescent probe for zinc ion

We report on a simple strategy for the determination of zinc ion by using surface-modified quantum dots. The probe consists of manganese-doped quantum dots made from zinc sulfide and capped N-acetyl-L-cysteine. The particles exhibit bright yellow-orange emission with a peak at 598?nm which can be attributed to the ⁴T₁→⁶A₁ transition of Mn(II). This bright fluorescence is effectively quenched by modifying the sulfur anion which suppresses the radiative recombination process. The emission of the probe can then be restored by adding Zn(II) which causes the formation of a ZnS passivation layer around the QDs. The fluorescence enhancement caused is linear in the 1.25 to 30?μM zinc concentration range, and the limit of detection is 0.67?μM.

Magnetic nanoparticles modified with polydimethylsiloxane and multi-walled carbon nanotubes for solid-phase extraction of fluoroquinolones

We have surface-functionalized magnetic particles (MPs) with polydimethylsiloxane and multi-walled carbon nanotubes in a two-step reaction. The MPs were applied to solid-phase extraction of the fluoroquinolones ofloxacin, norfloxacin, ciprofloxacin, enrofloxacin prior to their determination by capillary liquid chromatography. The effects of sample pH, adsorption time, type of eluent, desorption time and desorption temperature were investigated. Under the optimum conditions, the extraction efficiencies are in the range from 81.5?% to 94.1?%, with relative standard deviations (RSDs) of <7.6?%. The detection limits vary from 0.24 to 0.48?ng?mL^?1. The method was applied to the analysis of spiked mineral water and honey. The recoveries for the fluoroquinolones in the real samples range from 84.0?% to 112?%, with RSDs ranging from 2.9?% to 7.8?%.

An alternative easy method for antibody purification and analysis of protein–protein interaction using GST fusion proteins immobilized onto glutathione–agarose

Immobilization of small proteins designed to perform protein–protein assays can be a difficult task. Often, the modification of reactive residues necessary for the interaction between the immobilized protein and the matrix compromises the interaction between the protein and its target. In these cases, glutathione-S-transferase (GST) is a valuable tag providing a long arm that makes the bait protein accessible to the mobile flow phase of the chromatography. In the present report, we used a GST fusion version of the 8-kDa protein serine protease inhibitor Kazal-type 3 (SPINK3) as the bait to purify anti-SPINK3 antibodies from a rabbit crude serum. The protocol for immobilization of GST-SPINK3 to glutathione–agarose beads was modified from previously reported protocols by using an alternative bifunctional cross-linker (dithiobis(succinimidyl propionate)) in a very simple procedure and by using simple buffers under physiological conditions. We concluded that the immobilized protein remained bound to the column after elution with low pH, allowing the reuse of the column for alternative uses, such as screening for other protein–protein interactions using SPINK3 as the bait.

Simultaneous determination of four different antibiotic residues in honey by chemiluminescence multianalyte chip immunoassays

We are presenting the first method for identification and quantification of antibiotic derivatives in honey samples using regenerable antigen microarrays in combination with an automated flow injection system. The scheme is based on an indirect competitive immunoassay format using monoclonal antibodies bound to the surface of the microarray. The surface of glass slides was coated with epoxy-activated poly(ethylene glycol) and enables direct immobilization of the antibiotic derivatives. The antigen/antibody interaction on the surface of the chip can be detected by chemiluminescence (CL) read-out via CCD camera. The method allows for fast analysis of the four analytes simultaneously and without purification or extraction. An effective data evaluation method also was developed to warrant unambiguous identification of the spots and to establish grey levels of CL intensities. The software developed enables fast and automated processing of the CL images. Dose?Cresponse curves were obtained for the derivatives of enrofloxacin, sulfadiazine, sulfamethazine and streptomycin. Spiking experiments revealed adequate recoveries within the dynamic ranges of the calibration curves of enrofloxacin (92%?±?6%), sulfamethazine (130%?±?21%), sulfadiazine (89%?±?20%) and streptomycin (93%?±?4%).

Preconcentration of trace aluminum (III) ion using a nanometer-sized TiO2-silica composite modified with 4-aminophenylarsonic acid, and its determination by ICP-OES

We describe a nanometer sized composite material made from titanium dioxide and silica that was chemically modified with 4-aminophenylarsonic acid and used for selective solid-phase extraction, separation and preconcentration of of aluminum(III) prior to its determination by ICP-OES. Under optimized conditions, the static adsorption capacity is 56.58?mg?g^?1, the enrichment factor is 150, the relative standard deviation is 1.6% (for n?=?11), and the detection limit (3?s) is 60?pg?mL^?1. The method was validated by analyzing the reference materials GBW 09101 (hair) and GBW 10024 (scallop) and successfully applied to the determination of trace Al(III) in spiked water samples and human urine, with recoveries ranging from 96% to 101%.

A novel tin-bismuth alloy electrode for anodic stripping voltammetric determination of zinc

We report on a novel tin-bismuth alloy electrode (SnBiE) for the determination of trace concentrations of zinc ions by square-wave anodic stripping voltammetry without deoxygenation. The SnBiE has the advantages of easy fabrication and low cost, and does not require a pre-treatment (in terms of modification) prior to measurements. A study on the potential window of the electrode revealed a high hydrogen overvoltage though a limited anodic range due to the oxidation of tin. The effects of pH value, accumulation potential, and accumulation time were optimized with respect to the determination of trace zinc(II) at pH 5.0. The response of the SnBiE to zinc(II) ion is linear in the 0.5–25?μM concentration range. The detection limit is 50?nM (after 60?s of accumulation). The SnBiE was applied to the determination of zinc(II) in wines and honeys, and the results were consistent with those of AAS.

Neutron-Encoded Protein Quantification by Peptide Carbamylation

We describe a chemical tag for duplex proteome quantification using neutron encoding (NeuCode). The method utilizes the straightforward, efficient, and inexpensive carbamylation reaction. We demonstrate the utility of NeuCode carbamylation by accurately measuring quantitative ratios from tagged yeast lysates mixed in known ratios and by applying this method to quantify differential protein expression in mice fed a either control or high-fat diet.

Cyanide–Arene Meisenheimer Complex Generated in Electrospray Ionization Mass Spectrometry Using Acetonitrile as a Solvent

The C – C bond formation activated under negative electrospray ionization of an acetonitrile solution of 1,3,5-trinitrobenzene is reported. The solvent function is to provide a source of cyanide ion, a highly problematic reagent, which is found to attack the electron-deficient aromatic ring to form a covalently bound anionic complex (Meisenheimer complex). The structure of the complex is elucidated by means of collision induced dissociation mass spectrometry and IR multiple photon dissociation spectroscopy in the ‘fingerprint’ region.

Plasma-Assisted Reaction Chemical Ionization for Elemental Mass Spectrometry of Organohalogens

We present plasma-assisted reaction chemical ionization (PARCI) for elemental analysis of halogens in organic compounds. Organohalogens are broken down to simple halogen-containing molecules (e.g., HBr) in a helium microwave-induced plasma followed by negative mode chemical ionization (CI) in the afterglow region. The reagent ions for CI originate from penning ionization of gases (e.g., N₂) introduced into the afterglow region. The performance of PARCI-mass spectrometry (MS) is evaluated using flow injection analyses of organobromines, demonstrating 5–8 times better sensitivities compared with inductively coupled plasma MS. We show that compound-dependent sensitivities in PARCI-MS mainly arise from sample introduction biases.

Transferrin recognition based on a protein imprinted material prepared by hierarchical imprinting technique

A novel kind of transferrin imprinted polymer particles was synthesized by a hierarchical strategy. First, transferrin was immobilized on silica beads by non-covalent absorption. Then, a pre-polymerization mixture, composed of 1,4-bis(acryloyl)piperazine, methacrylamide, methacrylic acid, ammonium sulfate and polyoxyethylene sorbitan monolaurate, was irrigated into the pores of silica particles, and polymerized at 25 °C. Finally, the silica matrix was etched with ammonium hydrogen fluoride, not only to remove the template protein, but also to expose protein recognition sites on the surface of the imprinted polymer. The binding capacity of the transferrin-imprinted particles is 6.3 mg of protein per gram of material, and the time required to reach adsorption equilibrium was less than 10 min. The imprinting factor of transferrin is ca. 3.3 in the presence of ribonuclease B, cytochrome c and β-lactoglobulin. The results indicate that these imprinted polymer particles can recognize transferrin with good selectivity, high binding capacity and fast mass transfer. They may be applied as an artificial antibody to remove the high abundance proteins in plasma.

Comparison of three different configurations of dual ultramicroelectrodes for the decomposition of S-Nitroso-L-glutathione and the direct detection of nitric oxide

Podoplanin (PDP) is a small transmembrane protein and widely present in various specialized cells throughout the human body. It is a specific marker for identification of lymphatic vessel and a candidate marker for cancer stem cells in squamous cell carcinoma of the lung. We report on method for the highly selective determination of PDP by using a surface plasmon resonance imaging (SPRI) that exploits the highly selective interaction between PDP and anti-human PDP monoclonal antibody (IgG). The sensor has a dynamic range between 0.25 and 1.0?ng?mL^?1, and a detection limit of 15?pg?mL^?1. It was applied to the determination of PDP in blood plasma and tissue homogenates from paired normal and lung tumor tissue.

Single-drop microextraction for the determination of manganese in seafood and water samples

We describe a method for single drop microextraction of manganese from fish, mollusk, and from natural waters using the reagent 1-(2-pyridylazo)-2-naphthol as the complexing agent and chloroform as the fluid extractor. After extraction, the analyte was directly submitted to graphite furnace electrothermal atomic absorption spectrometry. Once optimized, the method has a detection limit of 30 ng L^?1, a limit of quantification of 100 ng L^?1, and an enrichment factor of 16. Its accuracy was verified by applying the procedure to the following certified reference materials: apple leaves, spinach leaves, bovine liver, and mussel tissue. The procedure was also successfully applied to the determination of manganese in seafood and natural waters.

Matrix Segregation as the Major Cause for Sample Inhomogeneity in MALDI Dried Droplet Spots

The segregation in dried droplet MALDI sample spots was analyzed with regard to the matrix-to-sample ratio using optical microscopy, MALDI imaging mass spectrometry (MALDI MSI) and IR imaging spectroscopy. In this context, different polymer/matrix/solvent systems usually applied in the analysis of synthetic polymers were investigated. The use of typical matrix concentrations (10 mg mL^?1) in almost every case resulted in ring patterns, whereas higher concentrated matrix solutions always led to homogeneous sample spot layers. The data revealed that segregation is predominantly caused by matrix transport in the drying droplet, whereas polymer segregation seems to be only secondary.

Carbon paste electrode modified with a binuclear manganese complex as a sensitive voltammetric sensor for tryptophan

We report on a carbon paste electrode that was modified with a binuclear manganese(II) complex by the drop-coating method. A study on the mechanism of the electro-oxidation of tryptophan (Trp) at this electrode indicated that it enables Trp to be determined with good sensitivity and selectivity. Second-order derivative linear sweep voltammetry at pH 4.1 revealed that a sensitive anodic peak appears at 812?mV (vs. SCE) whose current is proportional to the concentration of Trp in the concentration range from 0.1 to 1.0???mol?L^?1 and 1.0 to 80???mol?L^?1, with a detection limit (S/N?=?3) of 0.08???mol?L^?1 (60?s of accumulation). The method was applied to the determination of Trp in amino acid injection solutions with satisfactory results.

Functionalization of cross linked chitosan with 2-aminopyridine-3-carboxylic acid for solid phase extraction of cadmium and zinc ions and their determination by atomic absorption spectrometry

We have developed a new method for solid phase extraction (SPE) and preconcentration of trace amounts of cadmium and zinc using cross linked chitosan that was functionalized with 2-aminopyridine-3-carboxy acid. Analytical parameters, sample pH, effect of flow rate, sample volume, and concentration of eluent on column SPE were investigated. The effect of matrix ions on the recovery of cadmium and zinc has been investigated and were found not to interfere with preconcentration. Under the optimum experimental conditions, the preconcentration factors for Cd(II) and Zn(II) were found to be 90. The two elements were quantified via atomic absorption spectrometry. The detection limits for cadmium and zinc are 21 and 65?ng?L^?1, respectively. The method was evaluated by analyzing a certified reference material (NIST 1643e; water) and has been successfully applied to the analysis of cadmium and zinc in environmental water samples.

In-situ ionic liquid-dispersive liquid-liquid microextraction method to determine endocrine disrupting phenols in seawaters and industrial effluents

We have evaluated an in-situ ionic liquid-dispersive liquid-liquid microextraction procedure for the determination of six endocrine disrupting phenols in seawaters and industrial effluents using HPLC. The optimized method requires 38???L of the water-soluble ionic liquid 1-butyl-3-methylimidazolium chloride, and 5?mL of seawater or industrial effluent. After appropriate work-up, a drop (~10???L) of an ionic liquid is formed that contains the analytes of interest. It is diluted with acetonitrile and injected into the HPLC system. This procedure is accomplished without heating or cooling the solutions. The method is characterized by (a) average relative recoveries of 90.2%, (b) enrichment factors ranging from 140 to 989, and (c) precisions (expressed as relative standard deviations) of less than 11% when using a spiking level of 10?ng?mL^?1. The limits of detection range from 0.8?ng?mL^?1 for 4-cumylphenol to 4.8?ng?mL^?1 for bisphenol-A.

Profiling thiol metabolites and quantification of cellular glutathione using FT-ICR-MS spectrometry

We describe preparation and use of the quaternary ammonium-based α-iodoacetamide QDE and its isotopologue *QDE as reagents for chemoselective derivatization of cellular thiols. Direct addition of the reagents to live cells followed by adduct extraction into n-butanol and analysis by FT-ICR-MS provided a registry of matched isotope peaks from which molecular formulae of thiol metabolites were derived. Acidification to pH 4 during cell lysis and adduct formation further improves the chemoselectivity for thiol derivatization. Examination of A549 human lung adenocarcinoma cells using this approach revealed cysteine, cysteinylglycine, glutathione, and homocysteine as principal thiol metabolites as well as the sulfinic acid hypotaurine. The method is also readily applied to quantify the thiol metabolites, as demonstrated here by the quantification of both glutathione and glutathione disulfide in A549 cells at concentrations of 34.4?±?11.5 and 10.1?±?4.0 nmol/mg protein, respectively.