Qtrap 6500 lc ms ms system

For LC–MS/MS analysis, chromatographic separation was carried out on the HPLC–MS system Q-TRAP 6500 LC-MS/MS System from AB Sciex equipped with Shimadzu LC-20A LC and Auto Sampler system. The mixture was separated on a Synergi Fusion RP 4 μ 80°A from Phenomenex (150 × 2.00 mm). Tauro-conjugated and non-conjugated bile acids were separated at a flow rate of 200 μl/min using a methanol–aqueous ammonium acetate (NH₄OAc) gradient. Mobile phase A was water containing 5 mM ammonium acetate and 0.1% formic acid, mobile phase B was methanol, containing ammonium acetate at 5 mM and 0.1% formic acid. The gradient started at 65% B and increased to 85% B in 23 min, kept at 85% B for 5 min then decreased to 65% B in 1 min and kept at 65% B for 10 min. ESI was performed in negative ion mode, the ion source temperature was set at 280 °C. The tune page parameters were automatically optimized injecting taurocholic acid at 1 μM as standard.

The protocols used for the extraction and purification of DSF family components were described previously (12 (link)). L. enzymogenes strains were cultured in liquid medium for 24 h, and 50 ml of the bacterial supernatant was collected by centrifugation at 4000 × g and 4°C for 15 min. The pH of the supernatants was adjusted to 4.0 by adding hydrochloric acid prior to two extractions with an equal volume of ethyl acetate. Ethyl acetate fractions were collected, and the solvent was removed by rotary evaporation to dryness at 42°C. The residue was dissolved in 100 μl of methanol. Crude extract was subjected to 0.22-μm Mini-Star filtration, and the filtrate was concentrated to 100 μl for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The extract (2 μl) was injected into a C₁₈ reversed-phase column (2.1 × 100 mm; Agilent Poroshell SB-C₁₈) and eluted with water in methanol (20:80 [vol/vol]) at a flow rate of 0.3 ml/minute in a QTRAP 6500 LC-MS/MS system (AB Sciex, USA).

The Agilent series 1260 HPLC system was used for the analyses. The substances were separated on a Luna 3 µm Phenyl-Hexyl 150 mm × 2.0 mm column (Phenomenex, Torrance, NJ, USA) using water with 5 mM ammonium formate and acetonitrile as the mobile phase. The flow rate was 400 µL/min and the column was thermostated at 50 °C. Gradient elution was used. The injection volume was 2 µL, and the total LC analysis time was 40 min. Spectrometric analysis was performed using the AB Sciex QTRAP^® 6500 LC-MS/MS system (Framingham, MA, USA) with the Turbo Spray ion drive with positive ionization and positive and negative ionization. The spray voltage was set to 5000 V and −4500 V for positive and negative ionization, respectively. The source temperature was set to 550 °C. Nitrogen was used as the curtain gas (20 psi), collision gas (medium), and ion source gases, nebulizer gas (50 psi) and heating gas (55 psi). Analyst 1.6.2 software (AB Sciex, Framingham, MA, USA) was used to control the LC-MS/MS system and to archive the data.

Cultures were grown in LB medium at 28°C until OD₆₀₀, 1.5. Cells from 2 ml culture were harvested for protein quantification by the BCA assay (TransGen, China). Cells from 8 mL of culture were used for c-di-GMP extraction utilizing 0.6 M HClO₄ and 2.5 M K₂CO₃^{45 (link)}. Samples were assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis on an AB SCIEX QTRAP 6500 LC-MS/MS system^{41 (link)}.

Blood samples were collected from the caudal veins of cyp17a2^+/+ XY (n≥3) and cyp17a2^-/- XY (n≥3) fish after anesthesia (250 mg/L, MS-222, Sigma, St Louis, USA) at 180 dah and kept at room temperature for 1 h. After centrifugation at 3000 rpm for 10 min, the supernatant was transferred to a clean centrifuge tube. The supernatant was then centrifuged at 12000 rpm for 10 min at 4 °C and frozen at -80 °C until use. Serum steroid levels were measured using the EIA (enzyme-linked immunosorbent assay) kits (Cayman, Michigan, USA) or detected by MetWare (http://www.metware.cn/) based on the AB Sciex QTRAP^® 6500 LC-MS/MS system (AB Sciex, Framingham, USA). The steroid information was listed in Table 2.

An AB Sciex QTRAP6500 LC-MS/MS system was used to detect the content of phytohormones before and after pollination in the different pollination combinations reported earlier (Xiao et al., 2018 (link); López-Cristoffanini et al., 2019 (link)).

For LC-MS/MS analysis, chromatographic separation was carried out on the HPLC-MS system Q-TRAP 6500 LC-MS/MS System from AB Sciex equipped with Shimadzu LC-20A LC and AutoSampler system. The mixture was separated on a Synergi Fusion RP 4 m from Phenomenex (150 mm × 2.00 mm).
Tauro-conjugated and non-conjugated BAs were separated at a flow rate of 200 μL/min using a methanol–aqueous ammonium acetate (NH₄OAc) gradient. Mobile phase A was water containing 5 mM ammonium acetate and 0.1% formic acid, mobile phase B was methanol, containing ammonium acetate at 5 mM and 0.1% formic acid. The gradient started at 65% B and increased to 85% B in 23 min, kept at 85% B for 5 min then decreased to 65% B in 1 min and kept at 65% B for 10 min. ESI was performed in negative ion mode and the ion source temperature was set at 280°C. The tune page parameters were automatically optimized injecting taurocholic acid at 1 μM as standard. The MS/MS detection was operated in MRM mode using a collision energy of 20 (arbitrary units) and the observed transitions are reported in Mencarelli et al., 2013 (link).