Identification of disease- and nutrient-related metabolic fingerprints in osteoarthritic Guinea pigs.

Osteoarthritis (OA), one of the most common diseases among the elderly, is characterized by the progressive destruction of joint tissues. Its etiology is largely unclear and no effective disease-modifying treatment is currently available. Metabolic fingerprinting provides a novel tool for the identification of biomarkers.

A generic assay for phosphate-consuming or -releasing enzymes coupled on-line to liquid chromatography for lead finding in natural products.

A generic continuous-flow assay for phosphate-consuming or -releasing enzymes coupled on-line to liquid chromatography (LC) has been developed. Operating the LC-biochemical assay in combination with mass spectrometry allows the fast detection and identification of inhibitors of these enzymes in complex mixtures. The assay is based on the detection of phosphate, released by the on-line continuous-flow enzymatic reaction, using a fluorescent probe.

The stimulating effect of a harsh environment on the bacteriocin activity by Enterococcus faecium RZS C5 and dependency on the environmental stress factor used.

Bacteriocin production by Enterococcus faecium RZS C5 occurs in a growth-associated way but is generally switched off in the very early growth phase. The influence of environmental stress on the bacteriocin production kinetics by E. faecium RZS C5 was analysed at a controlled temperature of 35 degrees C and constant pH 6.

Establishment of the enzymatic protein acetylation independent of acetyl CoA: recombinant glutathione S-transferase 3-3 is acetylated by a novel membrane-bound transacetylase using 7,8-diacetoxy-4-methyl coumarin as the acetyl donor.

The current knowledge on biological protein acetylation is confined to acetyl CoA-dependent acetylation of protein catalyzed by specific acetyl transferases and the non-enzymatic acetylation of protein by acetylated xenobiotics such as aspirin. We have discovered a membrane-bound enzyme catalyzing the transfer of acetyl groups from the acetyl donor 7,8-diacetoxy-4-methyl coumarin (DAMC) to glutathione S-transferase 3-3 (GST3-3), termed DAMC:protein transacetylase (TAase). The purified enzyme was incubated with recombinant GST3-3 subunit and DAMC, the modified protein was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in gel digested with trypsin and the tryptic digest was analyzed by mass spectrometry.

Capillary electrophoretic separations of proteins using carrier ampholytes.

Capillary separations of proteins using carrier ampholytes are performed between an anolyte and a catholyte of same pH (pH 3). Depending upon the concentration of carrier ampholytes used, two different separation processes take place. At a 10% concentration, the high-resolution separation of six model proteins is achieved, which can be described as a transient capillary isoelectric focusing (cIEF) system moving isotachophoretically.

On-target fraction collection for the off-line coupling of capillary isoelectric focusing with matrix-assisted laser desorption/ionization mass spectrometry.

In the present work, we describe a collection system for the off-line coupling of capillary isoelectric focusing (CIEF) with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In this system, the capillary effluent is directly deposited in fractions onto the MALDI target via the use of a sheath liquid. The collected fractions are subsequently supplemented with matrix and further analysed by MALDI-TOF mass spectrometry for mass assignment.

Potential of on-line micro-LC immunochemical detection in the bioanalysis of cytokines.

An on-line liquid chromatography-immunochemical detection (LC-ICD) system for the quantification of cytokines in cell extracts has been developed using a post-column continuous-flow reaction detection system using fluorescence labelled antibodies. Cytokines eluting from the micro-HPLC column react with antibodies to form fluorescent complexes. In a second step the excess of free antibody is trapped on a cytokine bound support prior to fluorescence detection.

Analysis of histones by on-line capillary zone electrophoresis-electrospray ionisation mass spectrometry.

The on-line combination of capillary electrophoresis and electrospray ionisation mass spectrometry was applied for the determination of some basic histones from calf thymus. The separation performance of those basic proteins was significantly improved by coating the capillaries with hydroxypropylmethylcellulose. The coating appeared to mask effectively the underlying silanol groups thus avoiding undesirable adsorption of the histones onto the capillary walls, while it was also shown to be an effective way to avoid contamination of the mass spectrometer.

Urticaria and rhinitis to shrubs of Ficus benjamina and breadfruit in a banana-allergic road worker: evidence for a cross-sensitization between Moracea, banana and latex.

Background: We report the case of a road worker with a food allergy to banana, who developed urticaria and rhinitis when cutting shrubs of Ficus benjamina and breadfruit. He did not develop an allergy to latex of Hevea brasiliensis.

Results: Sensitization to latex of F.

In-line coupling of low-pressure short capillary electrochromatography columns to electrospray ionisation mass spectrometry.

A new in-house designed and constructed injection valve for capillary electrochromatography (CEC) based on a rotating injection part with compartments for the eluent as well as for the sample has been coupled to a mass spectrometer via a sheath flow electrospray ionisation (ESI) interface, using short capillary columns of 15 cm length. The CEC columns were packed with 3 microm C(18) bonded silica particles, and a mixture of peptides was analysed using an ammonium acetate/acetonitrile eluent. A significant increase in the signal-to-noise ratio was obtained when the peptides were dissolved in water with the same content of organic modifier as in the eluent with an addition of 0.

Separation and identification of platinum adducts with DNA nucleotides by capillary zone electrophoresis and capillary zone electrophoresis coupled to mass spectrometry.

Platinum adducts are supposed to be the cytotoxic lesions in DNA after platinum-containing anticancer therapy. Various adducts are formed upon interaction of platinum complexes with nucleotides, but contribution of individual adducts to antitumor activity and toxicity of platinum complexes still remains to be examined. A capillary zone electrophoresis (CZE) method is described that is suitable to separate individual platinum adducts.

Chip-based capillary electrophoresis with an electrodeless nanospray interface.

A sheathless and electrodeless nanospray interface has been used to interface a polycarbonate capillary electrophoresis (CE) chip to a mass spectrometer (MS). The chip was made of two flat polycarbonate plates which were bolted together. Channels were imprinted in one of the plates with metal wires, using a hydraulic press.

Free flow electrophoresis device for continuous on-line separation in analytical systems. An application in biochemical detection.

A free flow electrophoresis (FFE) device was developed for continuous electrophoretic separation of charged compounds and implemented in a continuous flow biochemical detection (BCD) system. These continuous separation characteristics make FFE well suitable for online implementation in a chromatographic or flow injection analysis system, in which an additional separation step of charged compounds is desired. In a heterogeneous biochemical flow assay for the determination of biotin, an analyte zone reacts with an excess of an affinity protein.

High-performance liquid chromatography coupled to enzyme-amplified biochemical detection for the analysis of hemoglobin after pre-column biotinylation.

The determination of proteins with enzyme-amplified biochemical detection (EA-BCD) coupled on-line with high-performance liquid chromatography (HPLC) is demonstrated. The EA-BCD system was developed to detect biotin-containing compounds. Hemoglobin, which was used as a model compound, was biotinylated prior to sample introduction.

Capillary electrochromatography/nanoelectrospray mass spectrometry for attomole characterization of peptides.

The successful coupling of capillary electrochromatography (CEC) to an ion trap mass spectrometer via a nanoelectrospray interface (nESI) is described. Using a conductively coated tip butted to the end of a CEC column, it was possible to obtain a stable spray without any sheath liquid being employed. Selected small peptides were separated with CEC columns (100 microm i.

A free-flow electrophoresis chip device for interfacing capillary isoelectric focusing on-line with electrospray mass spectrometry.

When electrospray ionisation mass spectrometry (ESI-MS) is used on-line with capillary isoelectric focusing (CIEF), the presence of the carrier ampholytes creating the IEF pH gradient is not desirable. With the purpose of removing these ampholytes, we have developed a free-flow electrophoresis (FFE) device and coupled it to CIEF. The different parameters inherent to the resulting CIEF/FFE system were optimised using ultraviolet absorbance (UV) detection.

On-line isotachophoretic sample focusing for loadability enhancement in capillary electrochromatography-mass spectrometry.

The use of isotachophoretic (ITP) sample focusing to improve the detection limits for the analysis of charged compounds in capillary electrochromatography (CEC) is described. A coupled-column set-up was used with a 220-microm inner diameter capillary, in which counterflow ITP focusing was performed, connected via a T-junction to a 75-microm inner diameter CEC capillary. As is illustrated, the use of ITP focusing resulted in a dramatic reduction of the sample concentration detection limits.

The application of HPLC with on-line coupled UV/MS-biochemical detection for isolation of an acetylcholinesterase inhibitor from narcissus 'Sir Winston Churchill'.

An HPLC with on-line coupled UV/MS-biochemical detection method for acetylcholinesterase (AChE) inhibitors in natural sources has been developed. The potential of this method is shown by the isolation of a new AChE inhibitor from the alcoholic extract of Narcissus 'Sir Winston Churchill'. Combining a prefractionation technique using centrifugal partition chromatography with the on-line HPLC-UV/MS-biochemical detection resulted in the isolation of the active compound that was identified as ungiminorine.

Pseudo-electrokinetic packing of high efficiency columns for capillary electrochromatography.

An improved and easy electrokinetic packing procedure is presented for the production of stable capillary columns suitable for capillary electrochromatography (CEC). In pseudo-electrokinetic packing a high electric field is used in conjunction with a hydrodynamic flow. The packing of silica-based reversed-phase columns can be achieved with basic, commercially available capillary electrophoresis (CE) equipment in approximately 15 min.

High-performance liquid chromatography with on-line coupled UV, mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products.

A high-performance liquid chromatography (HPLC) method with on-line coupled ultraviolet (UV), mass spectrometry (MS) and biochemical detection for acetylcholinesterase (AChE) inhibitory activity has been developed. By combining the separation power of HPLC, the high selectivity of biochemical detection, and the ability to provide molecular mass and structural information of MS, AChE inhibitors can be rapidly identified. The biochemical detection was based on a colorimetric method using Ellman's reagent.

Nanotechnology in bio/clinical analysis.

Nanotechnology is being exploited now in different fields of analytical chemistry: Single cell analysis; in chip/micro machined devices; hyphenated technology and sampling techniques. Secretory vesicles can be chemically and individually analyzed with a combination of optical trapping, capillary electrophoresis separation, and laser induced fluorescence detection. Attoliters (10(-18) l) can be introduced into the tapered inlets of separation capillaries.

Enantiomeric separation of clenbuterol by transient isotachophoresis-capillary zone electrophoresis-UV detection new optimization technique for transient isotachophoresis.

A method for the in-line preconcentration and enantioseparation of clenbuterol by transient isotachophoresis-capillary zone electrophoresis-UV absorbance detection (transient ITP-CZE-UV) has been developed. It implies the use of dimethyl-beta-cyclodextrin as chiral selector and the application of a hydrodynamic counterflow during the ITP step. ITP is used to focus the sample constituents prior to CE whereas a counterpressure counterbalances the electrophoretic migration of the compounds.

Enzyme amplification as detection tool in continuous-flow systems. II. On-line coupling of liquid chromatography to enzyme-amplified biochemical detection after pre-column derivatization with biotin.

Enzyme-amplified biochemical detection (EA-BCD) was used as a post-column detection technique, coupled on-line with high-performance liquid chromatography (HPLC). The enzyme detection system was developed to detect biotin or biotin containing compounds. Biotinylation is widely used to label analytes of interest ranging from small molecules to proteins and DNA.

Enzyme amplification as detection tool in continuous-flow systems. I. Development of an enzyme-amplified biochemical detection system coupled on-line to flow-injection analysis.

The on-line coupling of flow-injection analysis (FIA) to an enzyme-amplified biochemical detection (EA-BCD) system is described. The aim of this study is the development of a detection system able to detect biotin-containing compounds at low concentration levels. The detection system is based on the interaction of biotin with enzyme-labeled affinity proteins.