To explore the transglycosalating ability of Lcf, several acceptors, including methyl α-d -glucopyranoside, methyl β-d -glucopyranoside, glycerol, d -sorbitol, ethanol, and L-serine, were involved in the transglycosylation reactions. Briefly, Lcf (0.02 U) was added into the mixture containing laminarihexaose (2 mg/mL) and an acceptor (2 mg/mL) dissolved in sodium acetate buffer (25 mM, pH 5.6). Then, the resulting mixture (200 μL) was incubated at 37 °C for 5 min. The reaction was terminated by the addition of an equal volume of 2.5% (v/v) aqueous ammonia. After centrifugation, the supernatant was collected and detected using positive ion electrospray ionization mass spectrometry (ESI-MS) (Agilent 6460 Triple Quad, Santa Clara, CA, USA).
Agilent 6460 triple quad
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The Agilent 6460 Triple Quad is a highly sensitive and accurate mass spectrometry system designed for quantitative and qualitative analysis. It features a triple quadrupole configuration that enables precise detection and identification of a wide range of analytes.
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4 protocols using agilent 6460 triple quad
1
Enzymatic Transglycosylation of Acceptors
2
Quantification of Berberine in Tissues
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DIO mice were i.p injected with 5 mg/kg BBR. After 1 h, the animals were sacrificed and plasma, liver, different adipose depots, and muscle were collected and preserved at −80 °C. Tissue samples were analyzed with LC-MS/MS system (Agilent 1200 HPLC coupled to Agilent 6460 Triple Quad instrument, Agilent Technologies, USA) to detect the concentration of BBR. Samples were disrupted with methanol before analysis. The chemicals were firstly separated on Luna PFP (50 × 2.0 mm, 5 μm, Phenomenex) using mixture of methanol-0.1% formic acid and 5 mmol/L ammonium acetate solution (55:45, v/v) with following rate (0.65 ml/min, 0–5 min RT). The chemicals with m/z 336 and 320 were analyzed using electrospray ionization mode with capillary voltage setting to 4 kV. The drying gas temperature was 350 °C with a flow rate of 10 L/min, and the sheath gas temperature was 350 °C with a flow rate of 11 L/min. Data were analyzed by MassHunter Quantitative Analysis (version B.02.01, Agilent Technologies).
3
Quantification of A-769662 in Metabolic Tissues
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HFD-fed mice were i.p. injected with A-769662 (30 mg/kg). After 1 h, the animals were sacrificed, and the plasma, liver, and various adipose depots and muscles were collected and preserved at −80°C. Tissue samples were analyzed with a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) system (an Agilent 1200 HPLC coupled to an Agilent 6460 Triple Quad instrument, Agilent Technologies, USA) to detect the concentration of A-769662. Data were analyzed by MassHunter Quantitative Analysis (version B.02.01, Agilent Technologies, USA).
4
Quantification of Plasma Oxylipins
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For the detection of total plasma oxylipins, we took a plasma sample (200 μL), added 300 μL of 10M sodium hydroxide (NaOH), and subjected it to alkaline hydrolysis at 60 °C for 30 min. The sample pH was then adjusted to 6 using 300 μL 58% acetic acid. The prepared samples were then subjected to solid phase extraction (SPE) using a Varian Bond Elut Certify II column. Specific experimental steps were described as previously [11 (link),45 (link)]. For the detection of free plasma oxylipins, SPE extraction was performed directly after pH adjustment without prior alkaline hydrolysis.
The extracted metabolites were evaluated by LC-MS/MS using an Agilent 6460 Triple Quad mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) and an Agilent 1200 high-performance liquid chromatography (HPLC) system (degasser, binary pump, well plate sampler, thermostatic column chamber). A Phenomenex Kinetex column (150 mm 2.1 mm, 2.6 m; Phenomenex, Aschaffenburg, Germany) was used in the HPLC system. The specific analysis process has been described previously [45 (link)]. Free and total plasma oxylipins were measured in the blood samples.
The extracted metabolites were evaluated by LC-MS/MS using an Agilent 6460 Triple Quad mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) and an Agilent 1200 high-performance liquid chromatography (HPLC) system (degasser, binary pump, well plate sampler, thermostatic column chamber). A Phenomenex Kinetex column (150 mm 2.1 mm, 2.6 m; Phenomenex, Aschaffenburg, Germany) was used in the HPLC system. The specific analysis process has been described previously [45 (link)]. Free and total plasma oxylipins were measured in the blood samples.
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