Agilent 6420 triple quadrupole lc ms instrument

To detect 6-dehydroglucose reductase activity of MaSquF, SQ was reacted with MaSqoD as described above, and then MaSqoD was removed from the reaction mixture using a centrifugal filter unit (1.5 mL YM-30 Amicon, Millipore). To the 100 μL of the flow-through, 10 μM MaSquF and 0.5 mM NADPH were added, mixed and incubated at RT for 30 min. Negative controls were prepared by omitting SQ or MaSquF/MaSqoD. After the reaction, an equal volume of acetonitrile was added, followed by incubation for 10 min at 4°C, and the precipitated protein was removed by centrifugation (10,000 × g for 5 min at 4°C). The supernatant was filtered through a 0.22 μm nylon membrane filter prior to LC-MS analysis. LC-MS was performed on an Agilent 6420 Triple Quadrupole LC/MS instrument (Agilent Technologies). LC-MS analysis was carried out on a ZIC-HILIC column (5 mm, 200 Å, 150 × 4.6 mm; Merck). The HPLC conditions were as follows: from 90% B to 70% B in 10 min, 70% B to 50% B in 20 min, and 90% B for 5 min. Solvent A was 90% 0.1 M ammonium acetate and 10% acetonitrile, and solvent B was acetonitrile. The flow rate was set to 0.5 mL/min. The mass spectrometer was run in ESI negative mode.

A 200-μl reaction mixture containing 50 mM Tris-HCl, pH 7.5, 10 μM BwSauCD, 10 μM BwSauS, 1 mM ATP, 1 mM CoA, 1 mM NADPH, 20 mM sulfoacetate, 10 mM MgCl₂, 50 mM KCl was incubated at RT for 30 min. ATP, CoA, NADPH, sulfoacetate, BwSauCD, BwSauS was omitted in the negative controls. 30 μl of the reaction solution was mixed with 330 μl of 0.73 M sodium acetate buffer pH 5.0, followed by 240 μl of freshly prepared DNPH solution (10 mg dissolved in 25 ml methanol), and the mixture was incubated at 50 °C for 1 h and then filtered prior to LC-MS analysis. LC-MS analysis was performed on an Agilent 6420 Triple Quadrupole LC-MS instrument (Agilent Technologies). LC-MS analysis was carried out with 20 μl sample volume on an Agilent ZORBAX SB-C18 column (4.6 × 250 mm, product number 880975-902). The HPLC conditions were as follows: linear gradient from 15% to 85% solvent B in 20 min, where solvent A was 0.1% formic acid in H₂O, and solvent B was acetonitrile. The flow rate was set to 1 ml/min. The negative ion extracted ion chromatograms (EICs) for the product are shown in Figure 3, B and C.