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Small molecules containing carboxylic acid functional groups are ubiquitous throughout biology, playing vital roles in biological chemistry ranging from energy metabolism to cellular signaling. This paper describes a new derivatization reagent, 4-bromo- N-methylbenzylamine, which was selected for its potential to derivatize mono-, di- and tri-carboxylic acids, such as the intermediates of the tricarboxylic acid (TCA) cycle. This derivatization procedure facilitated the use of positive electrospray ionization (ESI) tandem mass spectrometry (MS/MS) detection of derivatized species allowing for clear identification thanks to the easily recognizable isotope pattern of the incorporated bromine. A liquid chromatography (LC)-MS/MS method was developed which provided limits of detection between 0.2 and 44 μg L in under 6 min, depending on the analyte and total analysis time. This method was successfully applied in both in vitro and in vivo models. [ABSTRACT FROM AUTHOR]

A matrix removal procedure with ion-exchange resin prior to analysis for 18 fluorinated benzoic acids (FBAs) tracers in saline (>25% salt) reservoir water was optimized. The elimination of >98% of salt and the simultaneous matrix sample cleanup allowed the direct analysis using the supernatant by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This resulted in a gain in detection limits for most of the tracers in comparison with the reference method (direct analysis after minimum required dilution). The limits of detection (LODs) were in the range of 0.01-0.15 ng/ml and compared to other studies the developed method provided comparable limits of detection and advantage of simplified and shorter sample preparation. The presented method offers a considerable gain in simplicity and analysis time. Recoveries for all the tracers reached 80-100%, except for 2-FBA and 2,6-dFBA for which they were ca. 60%. The low recoveries were corrected by the use of five isotopically labeled internal standards. The method was validated by the analysis of spiked samples and by an independent comparison of the results with those obtained by solid-phase extraction LC-MS/MS method. [ABSTRACT FROM AUTHOR]

Chronic post-surgical pain (CPSP) is a normal and significant symptom in clinical surgery, such as breast operation, biliary tract operation, cesarean operation, uterectomy and thoracic operation. Severe chronic post-surgical pain could increase post-surgical complications, including myocardial ischemia, respiratory insufficiency, pneumonia and thromboembolism. However, the underlying mechanism is still unknown. Herein, a rat CPSP model was produced via thoracotomy. After surgery, in an initial study, 5 out of 12 rats after surgery showed a significant decrease in mechanical withdrawal threshold and/or increase in the number of acetone-evoked responses, and therefore classified as the CPSP group. The remaining seven animals were classified as non-CPSP. Subsequently, open-chest operation was performed on another 30 rats and divided into CPSP and non-CPSP groups after 21-day observation. Protein expression levels in the dorsal spinal cord tissue were determined by 12.5 % SDS-PAGE. Finally, differently expressed proteins were identified by LC MS/MS and analyzed by MASCOT software, followed by Gene Ontology cluster analysis using PANTHER software. Compared with the non-CPSP group, 24 proteins were only expressed in the CPSP group and another 23 proteins expressed differentially between CPSP and non-CPSP group. Western blot further confirmed that the expression of glutaminase 1 (GLS1) was significantly higher in the CPSP than in the non-CPSP group. This study provided a new strategy to identify the spinal proteins, which may contribute to the development of chronic pain using differential proteomics, and suggested that GLS1 may serve as a potential biomarker for CPSP. [ABSTRACT FROM AUTHOR]

The paper presents the development of an advanced extraction and fast analytical LC MS/MS method for simultaneous analyses of reduced and oxidized glutathione (GSH and GSSG, respectively) in different animal tissues. The simultaneous determination of GSH and GSSG is crucial because the amount and ratio of both GSH and GSSG may be altered in response to oxidative stress, an important mechanism of toxicity. The method uses the derivatization of free thiol groups in GSH. Its performance was demonstrated for less explored tissues (lung, brain, and liver) in mouse. The combined extraction and analytical method has very low variability and good reproducibility, maximum coefficients of variance for within-run and between-run analyses under 8 %, and low limits of quantification; for GSH and GSSG, these were 0.2 nM (0.06 ng/mL) and 10 nM (6 ng/mL), respectively. The performance of the method was further demonstrated in a model experiment addressing changes in GSH and GSSG concentrations in lung of mice exposed to CdO nanoparticles during acute 72 h and chronic 13-week exposures. Inhalation exposure led to increased GSH concentrations in lung. GSSG levels were in general not affected; nonsignificant suppression occurred only after the longer 13-week period of exposure. The developed method for the sensitive detection of both GSH and GSSG in very low tissue mass enables these parameters to be studied in cases where only a little sample is available, i.e. in small organisms or in small amounts of tissue. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the possible effect of pulse-modulated radiofrequency radiation (RFR) on rat skin hydroxyproline content, since skin is the first target of external electromagnetic fields. Skin hydroxyproline content was measured using liquid chromatography mass spectrometer method. Two months old male wistar rats were exposed to a 900 MHz pulse-modulated RFR at an average whole body specific absorption rate (SAR) of 1.35 W/kg for 20 min/day for 3 weeks. The radiofrequency (RF) signals were pulse modulated by rectangular pulses with a repetition frequency of 217 Hz and a duty cycle of 1:8 (pulse width 0.576 ms). A skin biopsy was taken at the upper part of the abdominal costa after the exposure. The data indicated that whole body exposure to a pulse-modulated RF radiation that is similar to that emitted by the global system for mobile communications (GSM) mobile phones caused a statistically significant increase in the skin hydroxyproline level ( p = 0.049, Mann-Whitney U test). Under our experimental conditions, at a SAR less than the International Commission on Non-Ionizing Radiation Protection safety limit recommendation, there was evidence that GSM signals could alter hydroxyproline concentration in the rat skin. [ABSTRACT FROM AUTHOR]

In this study, three methods of protein extraction from the seeds of the Chinese fir were compared by examining the quality (including the number of protein spots observed) in the two-dimensional gel electrophoresis (2-DE), obtained by isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis of the total released protein. Three protein extraction methods were: TCA-acetone precipitation, SDS extraction/acetone precipitation, and phenol extraction methanol/ammonium acetate precipitation. The results showed that TCA-acetone precipitation was the most effective method for protein extraction; it gave the highest yield of total protein (8.9 mg protein per g seed weight) and the greatest number of proteins spots (1,034 spots) on the 2-DE gel. Further, several proteins were identified by liquid chromatography mass spectrometry (LC MS/MS), which are legumin-like storage protein, similar to AMP binding/acetate-CoA ligase, similar to 40S ribosomal protein S20, actin, ascorbate peroxidase, Similar to cysteine synthase, and unknown protein. These data demonstrates that TCA-acetone precipitation followed by 2-DE and LC MS/MS is a suitable method for proteomic analysis of coniferous species, such as Chinese fir and provides a valuable starting point for similar proteomic analysis of other coniferous tree species. [ABSTRACT FROM AUTHOR]

With the genomes of three Frankia strains available, high-throughput proteomics methods can be used to reveal the set of proteins expressed by these bacteria in symbiosis with plants. A question we address is the degree to which the known genomes can be used to study proteomes of uncharacterized frankiae growing in field-collected root nodules. To this end, we have characterized the symbiotic proteomes of Frankia from three plant species, Alnus incana subsp. rugosa, Ceanothus americanus, and Elaeagnus angustifolia. Root nodule proteins were identified using two-dimensional liquid chromatography coupled to tandem mass spectrometry (LC MS/MS) of trypsin-digested protein samples. We identified 1300 Frankia proteins in A. incana nodules using the Frankia alni ACN14a genome and 1100 proteins from E. angustifolia nodules using the EAN1pec genome . In addition, over 100 proteins were identified from C. americanus nodules using a more limited one dimensional LC MS/MS analysis . Many of the most abundant proteins identified are involved in energy and nitrogen metabolism. The enzyme nitrogenase and the nitrogenase iron protein were among the most abundant proteins, reflecting the major process occurring in symbiosis. Several hundred plant proteins were also identified. We highlight the power of proteomics to uncover the physiology of symbiotic Frankia in the environment using heterologous genome information. [ABSTRACT FROM AUTHOR]