Orbitrap q exactive series mass spectrometer

We used a Vanquish UHPLC system (Thermo Fisher, 100 × 2.1 mm, 1.9 μm) to perform chromatographic separation of the samples at a constant temperature of 40°C and an Orbitrap Q Exactive series mass spectrometer (Thermo Fisher) to detect eluted metabolites. Specific type of column used in the UHPLC-MS/MS analysis was C18. The sample injection volume was 5 μl, and the column flow rate was maintained at 0.2 mL/min. The mobile phase contained two solvent eluents. In positive mode, eluent A was 0.1% FA in water, and eluent B was methanol; in negative mode, eluent A was 5 mM ammonium acetate with a pH of 9.0, and eluent B was methanol. The gradient elution was 2% B for 1.5 min, 2-100% B for 12.0 min, 100% B for 14.0 min, 100-2% B for 14.1 min, and 2% B for 17 min. To analyze the samples, we set the mass spectrometer spray voltage to 3.2 kV, the capillary temperature to 320°C, the sheath gas flow rate to 35 arb, and the auxiliary gas flow rate to 10 arb.

Liquid chromatography–tandem mass spectrometry analyses were performed using the Vanquish UHPLC system (Thermo Fisher Scientific, Shanghai, China) coupled with the Orbitrap Q Exactive series mass spectrometer (Thermo Fisher Scientific, Shanghai, China). A Hypersil Gold HPLC column (Thermo Fisher Scientific; 100 mm × 2.1 mm, 1.9 μm) with a 16-min linear gradient and 0.2 ml/min flow rate was employed. Eluent A (0.1% formic acid in water) and eluent B (methanol) were used for positive polarity mode. Eluent A (5 mM ammonium acetate, pH 9.0) and eluent B (methanol) were used for negative polarity mode. The following solvent gradient was used: 2% eluent B, 1.5 min; 2–100% eluent B, 12.0 min; 100% eluent B, 14.0 min; 100–2% eluent B, 14.1 min; and 2% eluent B, 17 min. The mass spectrometer was operated in positive/negative polarity mode with the following settings: spray voltage = 3.2 kV, capillary temperature = 320°C, sheath gas flow rate = 35 arbitrary unit (arb), and auxiliary gas flow rate = 10 arb.

Samples from C. pubescens and C. chinense were collected after 0 h and 6 h of cold stress for metabolic profiling. Next, 100 mg of dry tissue was powdered in liquid nitrogen, and the resulting powder was then suspended in 80% methanol and 0.1% formic acid using a vortex. The tissue samples were incubated on ice for 5 min, then subjected to centrifugation at 15,000 RPM at 4 °C for 5 min. To prepare the diluted supernatant, the suspension was diluted to a final concentration of 60% with LC-MS-grade water. The material was then transferred to a 0.22 μm filtered Eppendorf tube and subjected to centrifugation at 15,000× g at 4 °C for 10 min. In the last step, the filtrate was injected into the LC-MS/MS apparatus for analysis. A higher resolution LC-MS/MS coupled with an Orbitrap Q Exactive series mass spectrometer (Thermo Fisher, Newton Drive, Carlsbad, CA, USA) and a Vanquish UHPLC system (Thermo Fisher) was used.

Metabolite extracts were analyzed using a Vanquish UHPLC system (Thermo Fisher, United States) coupled with an Orbitrap Q Exactive series mass spectrometer (Thermo Fisher, United States). The raw data files generated by UHPLC-MS/MS were processed using the Compound Discoverer 3.1 (CD3.1, Thermo Fisher) to perform peak alignment, peak picking, and quantitation for each metabolite. 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 mzCloud¹ and ChemSpider² database to obtained the accurate qualitative and relative quantitative results. We applied univariate analysis (t-test) to calculate the statistical significance (P-value). The metabolites with VIP > 1 and P-value < 0.05 and fold change ≥ 2 or FC ≤ 0.5 were considered to be differential metabolites. The functions of metabolites and metabolic pathways were studied using the KEGG database. The reproducibility of untargeted analysis was assessed using six biological replicates.

LC-MS/MS analysis was operated in both positive and negative ion modes with the parameters optimized according to previously published procedures with some modifications (17 (link)). LC-MS/MS analyses were performed using a Vanquish UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive series mass spectrometer (Thermo Fisher). Samples were injected into the Hyperil Gold column (100 × 2.1 mm, 1.9 μm) using a 16-min linear gradient at a flow rate of 0.2 mL/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 mM ammonium acetate, pH 9.0) and eluent B (Methanol). The solvent gradient was set as follows: 1.5 min, 2% B; 12.0 min, 100% B; 14.0 min, 100% B; 14.1 min, 2% B; 16 min, 2% B. The flow rate was 0.2 mL/min. Q Exactive mass series spectrometer was operated in positive/negative polarity mode with a spray voltage of 3.2 kV, a capillary temperature of 320°C, a sheath gas flow rate of 35 arb, and an aux gas flow rate of 10 arb.

Samples of roots and leaves from each treatment group were collected for RNA-seq assays. Every treatment had four biological replicates. The extraction of metabolites was performed according to the procedure described by Ma et al. (2019) [8 ]. Sample tissues (0.1 g) were ground to a powder with liquid nitrogen. Liquid chromatography-mass spectrometry (LC–MS) analyses were performed using a Vanquish UHPLC system (Thermo Fisher, USA) coupled with an Orbitrap Q Exactive series mass spectrometer (Thermo Fisher, USA) (Novogene Bioinformatics Institute, Beijing, China). Compound Discoverer 3.1 (CD3.1, Thermo Fisher) was used to perform peak alignment, peak selection, and quantitation for each metabolite. Then, the peaks were matched with the mzCloud (https://www.mzcloud.org/) mzVault and MassList databases 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). The metabolites with VIP > 1, P < 0.05 and |log2FoldChange| ≥ 2 or FC ≤ 0.5 were regarded as differential metabolites. Pearson’s correlation analysis was used to integrate metabolome and transcriptome analyses.