Schizophrenia is a chronic disease characterized by the impairment of mental functions with a marked social dysfunction. A quantitative proteomic approach using iTRAQ labeling and SRM, applied to the characterization of mitochondria (MIT), crude nuclear fraction (NUC), and cytoplasm (CYT), can allow the observation of dynamic changes in cell compartments providing valuable insights concerning schizophrenia physiopathology. Mass spectrometry analyses of the orbitofrontal cortex from 12 schizophrenia patients and 8 healthy controls identified 655 protein groups in the MIT fraction, 1500 in NUC, and 1591 in CYT. We found 166 groups of proteins dysregulated among all enriched cellular fractions. Through the quantitative proteomic analysis, we detect as the main biological pathways those related to calcium and glutamate imbalance, cell signaling disruption of CREB activation, axon guidance, and proteins involved in the activation of NF-kB signaling along with the increase of complement protein C3. Based on our data analysis, we suggest the activation of NF-kB as a possible pathway that links the deregulation of glutamate, calcium, apoptosis, and the activation of the immune system in schizophrenia patients. All MS data are available in the ProteomeXchange Repository under the identifier PXD015356 and PXD014350.

The number of substances nominally listed in the prohibited list of the World Anti-Doping Agency increases each year. Moreover, many of these substances do not have a single analytical target and must be monitored through different metabolites, artifacts, degradation products, or biomarkers. A new analytical method was developed and validated for the simultaneous analysis of peptides and organic molecules using a single sample preparation and LC-Q-HRMS detection. The simultaneous analysis of 450 target molecules was performed after cleanup on a mixed-mode solid-phase extraction cartridge, combined with untreated urine. The cleanup solvent and reconstitution solvent were the most important parameters for achieving a comprehensive sample preparation approach. A fast chromatographic run based on a multistep gradient was optimized under different flows; the detection of all substances without isomeric coelution was achieved in 11 minutes, and the chromatographic resolution was considered a critical parameter, even in high-resolution mass spectrometry detection. The mass spectrometer was set to operate by switching between positive and negative ionization mode for FULL-MS, all-ion fragmentation, and FULL-MS/MS . The suitable parameters for the curved linear trap (c-trap) conditions were determined and found to be the most important factors for the development of the method. Only FULL-MS/MS enables the detection of steroids and peptides at concentrations lower than the minimum required performance levels set by World Anti-Doping Agency (1 ng mL ). The combination of the maximum injection time of the ions into the c-trap, multiplexing experiments, and loop count under optimized conditions enabled the method to be applied to over 10 000 samples in only 2 months during the 2016 Rio Summer Olympic and Paralympic Games. The procedure details all aspects, from sample preparation to mass spectrometry detection. FULL-MS data acquisition is performed in positive and negative ion mode simultaneously and can be applied to untargeted approaches.

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Food intake in fish and mammals is orchestrated by hypothalamic crosstalk between orexigenic (food intake stimulation) and anorexigenic (food intake inhibition) signals. Some of these signals are released by peripheral tissues that are associated with energy homeostasis or nutrient availability. During the fish larva stage, orexigenic stimulation plays a critical role in individual viability. The goal of this study was to assess the mRNA levels of the main neuropeptides involved in food intake regulation (npy, agrp, carppt, and pomc), in concert with the mRNA levels and peptide levels of ghrelin, under a fasting intervention at the larval stage in zebrafish (Danio rerio). Prior to the fasting intervention, the zebrafish larva cohort was reared for 20 days post fertilization (dpf) and then randomly divided into two groups of 20 individuals. One group was subjected to a fasting intervention for 5 days (fasted group), and the other group was fed normally (fed group); this experimental protocol was performed twice independently. At the end of the fasting period, individuals from each experimental group were divided into different analysis groups, for evaluations such as relative gene expression, immunohistochemistry, and liquid chromatography coupled to nano high-resolution mass spectrometry (nLC-HRMS) analyses. The relative expression levels of the following genes were assessed: neuropeptide Y (npy), agouti-related peptide (agrp), proopiomelanocortin (pomc), cocaine and amphetamine-regulated transcript (cartpt), ghrelin (ghrl), ghrelin O-acyltransferase (mboat4), growth hormone secretagogue receptor (ghsr), and glucokinase (gck). In the fasted group, significant upregulation of orexigenic peptides (npy – agrp) and ghsr was observed, which was associated with significant downregulation of gck. The anorexigenic peptides (pomc and cartpt) did not show any significant modulation between the groups, similar to mboat4. Contrary to what was expected, the relative mRNA upregulation of the orexigenic peptides observed in the fasted experimental group could not be associated with significant ghrelin modulation as assessed by three different approaches: qPCR (relative gene expression of ghrelin), nLC-HRMS (des-acyl-ghrelin levels), and immunohistochemistry (integrated optical density of prepropeptides in intestinal and hepatopancreas tissues). Our results demonstrate that zebrafish larvae at 25 dpf exhibit suitable modulation of the relative mRNA levels of orexigenic peptides (npy and agrp) in response to fasting intervention; nevertheless, ghrelin was not coregulated by fasting. Therefore, it can be suggested that ghrelin is not an essential peptide for an increase in appetite in the zebrafish larva stage. These results give rise to new questions about food intake regulation factors in the early stages of fish.

Zebrafish (Danio rerio) water tank (ZWT) approach was investigated as an alternative model for metabolism studies based on six different experiments with four model compounds. Sibutramine was applied for the multivariate optimization of ZWT conditions, also for the comparison of the metabolism among ZWT, humans and mice, beyond for the role of CYP2B6 in ZWT. After the optimization, 18 fish and 168 hours of experiments is the minimum requirement for a relevant panel of biotransformation products. A comparison among the species resulted in the observation of the same hydroxylated metabolites, with differences in metabolites concentration ratio. However, the ZWT allowed tuning of the conditions to obtain a specific metabolic profile, depending on the need. In addition, by utilizing CYP2B6 inhibition, a relevant ZWT pathway for the demethylation of drugs was determined. The stereospecificity of the ZWT metabolism was investigated using selegiline and no racemization or inversion transformations were observed. Moreover, the investigation of metabolism of cannabimimetics was performed using JWH-073 and the metabolites observed are the same described for humans, except for the hydroxylation at the indol group, which was explained by the absence of CYP2C9 orthologs in zebrafish. Finally, hexarelin was used as a model to evaluate studies by ZWT for drugs with low stability.As a result, hexarelin displays a very fast metabolization in ZWT conditions and all the metabolites described for human were observed in ZWT. Therefore, the appropriate conditions, merits, and relevant limitations to conduct ZWT experiments for the investigation of drug metabolism are described.

Metabolomics is an emerging technology that is increasing both in basic science and in human applications, providing a physiological snapshot. It has been highlighted as one of the most wide ranging and reliable tools for the investigation of physiological status, the discovery of new biomarkers and the analysis of metabolic pathways. Metabolomics uses innovative mass spectrometry (MS) allied to chromatography or nuclear magnetic resonance (NMR). The recent advances in bioinformatics, databases and statistics, have provided a unique perception of metabolites interaction and the dynamics of metabolic pathways at a system level. In this context, several studies have applied metabolomics in physiology- and disease-related works. The application of metabolomics includes, physiological and metabolic evaluation/monitoring, individual response to different exercise, nutritional interventions, pathological processes, responses to pharmacological interventions, biomarker discovery and monitoring for distinct aspects, such as: physiological capacity, fatigue/recovery and aging among other applications. For metabolomic analyses, despite huge improvements in the field, several complex methodological steps must be taken into consideration. In this regard, the present article aims to summarize the novel aspects of metabolomics and provide a guide for metabolomics for professionals related to physiologist and medical applications.

Ephedrines are widely used in therapy. Because of their stimulant properties, these substances are relevant in different forensic fields. At present, the state of the art for ephedrines quantification relay based on a liquid chromatography mass spectrometry, mainly because of the dilute-andshoot approach. Notwithstanding, several gas chromatography based methods have already been described, all of them include cleanup steps, with the potential disadvantage of incurring errors and increasing the workload. In this paper, a straightforward method for ephedrine quantification based on gas chromatographic mass spectrometry, without cleanup and based on Doehlert matrix optimization is presented. Only 10 µL of a urine sample is necessary and for N-methyl-N-(trimethylsilyl)trifluoroacetamide/N-methyl-bis-trifluoracetamide derivatives, the intermediate precision was 2.77% for ephedrine, 9.20% for cathine, 8.29% for norephedrine and 4.27% for pseudoephedrine. The limit of detection was 20 ng mL -1 for ephedrine, 30 ng mL -1 for cathine and 40 ng mL -1 for norephedrine and pseudoephedrine.

Background Casbene synthase (CS) is responsible for the first committed step in the biosynthesis of phorbol esters (PE) in the Euphorbiaceae. PE are abundant in the seeds of the biofuel crop Jatropha curcas and its toxicity precludes the use of the protein-rich cake obtained after oil extraction as an animal feed and the toxicity of the fumes derived from burning PE containing biofuel is also a matter of concern. This toxicity is a major hindrance to exploit the potential of this crop as a source of raw material to produce biodiesel. For this reason, the current research on J. curcas is mainly focused on the understanding of the biosynthesis and site of synthesis of PE, as an avenue for the development of genotypes unable to synthesize PE in its seeds. Results Here, we present targeted proteomics assays (SRM and PRM) to detect and quantify CS in leaves, endosperm, and roots of two J. curcas genotypes with contrasting levels of PE. These assays were based on the use of reference isotopic labeled synthetic peptides (ILSP) predicted from 12 gene models of CS from the J. curcas genome. Conclusion Our targeted proteomics methods were able to detect and quantify, for the first time, CS gene products and demonstrate the distribution of CS isoforms only in roots from J. curcas genotypes with a high and low concentration of PE. These methods can be expanded to monitor CS, at the protein level, in different tissues and genotypes of J. curcas.