Agilent 1100 series lc msd xct plus ion trap mass spectrometer

Yeast transformants were grown in 3 ml SC-Trp-Ura dropout media for two days. Culture broth was used directly for HPLC-ESI-MS analysis. HPLC-ESI-MS was performed on an Agilent 1100 series LC/MSD XCT plus ion trap mass spectrometer (Agilent, Palo Alto, CA) with a reverse-phase kinetex C18 column (Phenomenex, Torrance, CA). HPLC parameters for detection of compounds 4 and 5 were as follows: solvent A, 0.1% formic acid in water; solvent B, 0.1% formic acid in acetonitrile; gradient, 0% B to 100% B in 20 min, maintain at 100% B for 10 min, return and maintain at 10% B for 7 min; flow rate 0.3 ml/min; detection by UV spectroscopy at 300 nm or 330nm. Under such conditions, compounds 4 and 5 are eluted at 13.1 min and 15.6 min, respectively. Mass spectra were acquired in ultra scan mode using electrospray ionization (ESI) with positive/negative polarity. The MS system was operated using a drying temperature of 350 °C, a nebulizer pressure of 35 psi, a drying gas flow of 9 l/min, and a capillary voltage of 4500 V.

For in vitro enzymatic assays, the final concentrations of enzymes were 1 μM, 1 mM 5-MOA as substrate, with cofactor concentrations as 2 mM NADPH, 10 mM ATP and 10 mM MgCl₂. The assays were carried out in 50 mM Tris-HCl pH 8.5 buffer. The reaction mixtures were incubated at room temperature.
A typical volume of the reaction is 200 μl. 20 μl of reaction mixture was taken out at various time points and quenched with HCl. Each reaction mixture was used for HPLC-ESI-MS analysis. HPLC-ESI-MS was performed on an Agilent 1100 series LC/MSD XCT plus ion trap mass spectrometer (Agilent, Palo Alto, CA) with a reverse-phase kinetex C18 column (Phenomenex, Torrance, CA). HPLC parameters for detection of compound 5 were as follows: solvent A, 0.1% formic acid in water; solvent B, 0.1% formic acid in acetonitrile; gradient, 0% B to 100% B in 20 min, maintain at 100% B for 10 min, return and maintain at 10% B for 7 min; flow rate 0.3 ml/min; detection by UV spectroscopy at 330 nm. The amount of product 5 was quantified by area integration of the UV peak at 330 nm. A standard curve was generated using isolated compound 5 with the same HPLC condition.