metabolomics of zebrafish at different temperatures
Temperature has a significant impact on the feeding, reproduction, and metabolism of organisms. The metabolome refers to the endogenous small molecules that make up the metabolic components in an organism's cells, tissues, and organs. The complex metabolic activities and their regulation in an organism play a crucial role in its growth, development, immunity, and other aspects. Metabolites, which are the downstream end-products of gene and protein expression, serve as endpoints for bioinformation transmission and can provide a comprehensive reflection of the organism's physiological and pathological state. In this study, we conducted zebrafish culturing experiments at different water temperatures (HT, 28 ℃/LT, 16 ℃) and employed high-throughput liquid chromatography-mass spectrometry (LC/MS) to investigate the metabolic profiles of the zebrafish gut. The results revealed significant differences in metabolites at different temperatures, and these differences were strongly associated with changes in gut microbes.
Steps to reproduce
1. Metabolites Extraction Zebrafish were cultured at different water temperatures (HT, 28 ℃/LT, 16 ℃), intestines were collected to investigate the metabolic profile. Intestines (100 mg) were individually grounded with liquid nitrogen and the homogenate was resuspended with prechilled 80% methanol by well vortex. The sampleswere incubated on ice for 5 min and then were centrifuged at 15,000 g, 4°C for 20 min. Some of supernatant was diluted to final concentration containing 53% methanol by LC-MS grade water.The samples were subsequently transferred to a fresh Eppendorf tube and thenwere centrifuged at 15000 g, 4°C for 20 min. Finally, the supernatant was injected into the LC-MS/MS system analysis. 2. UHPLC-MS/MS Analysis UHPLC-MS/MS analyses were performed using a Vanquish UHPLC system (ThermoFisher, Germany) coupled with an Orbitrap Q ExactiveTMHF-X mass spectrometer (Thermo Fisher，Germany) in Novogene Co., Ltd. (Beijing, China). Samples were injected onto a Hypesil Gold column (100×2.1 mm, 1.9μm) using a 17-min linear gradient at a flow rate of 0.2mL/min. The eluents for the positive polarity mode were eluent A (0.1% FA in Water) and eluent B (Methanol).The eluents for the negative polarity mode were eluent A (5 mMammonium acetate, pH 9.0) and eluent B (Methanol).The solvent gradient was set as follows: 2% B, 1.5 min; 2-100% B, 12.0 min; 100% B, 14.0 min；100-2% B, 14.1 min；2% B, 17 min. Q ExactiveTM HF mass spectrometer was operated in positive/negative polarity mode with spray voltage of 3.2 kV, capillary temperature of 320°C, sheath gas flow rate of 40 arb and aux gas flow rate of 10 arb, Funnel RF level of 40, Aux gas heater temperature of 350°C. 3. Data processing and metabolite identification The raw data files generated by UHPLC-MS/MS were processed using the Compound Discoverer 3.1 (CD3.1, ThermoFisher) to perform peak alignment, peak picking, and quantitation for each metabolite. The main parameterswere set as follows: retention time tolerance, 0.2 minutes; actual mass tolerance, 5ppm; signal intensity tolerance, 30%; signal/noise ratio, 3; and minimum intensity, et al. After that, peak intensities were normalized to the total spectral intensity.The normalized data was used to predict the molecular formula based on additive ions, molecular ion peaks and fragment ions. And then peaks were matched with the mzCloud (https://www.mzcloud.org/)，mzVault and MassList database to obtain the accurate qualitative and relative quantitative results.Statistical analyses were performed using the statistical software R (R version R-3.4.3),Python (Python 2.7.6 version) and CentOS (CentOS release 6.6),When data were not normally distributed, normal transformations were attempted using of area normalization method 4. Data Analysis These metabolites were annotated using the KEGG database (https://www.genome.jp/kegg/pathway.html) ，HMDB database (https://hmdb.ca/metabolites) and LIPIDMaps database (http://www.lipidmaps.org/).