Microq tof 2 mass spectrometer

HPLC–MS was performed on an Agilent 1260 system with 1260 Infinity UV detector and a Bruker microQ-TOF II mass spectrometer equipped with an ESI ionization probe. The innoval C18 column (4.6 × 250 mm; 5 μm particle size) was used in this study. The HPLC conditions for isovaleryl-CoA, HIBP and PIVP were as follows: solvent A = H₂O (containing 20 mM ammonium acetate for the analysis of isovaleryl-CoA, or containing 0.1 % formic acid for the analysis of HIBP and PIVP); solvent B = methanol; flow rate = 1 mL/min; 0–5 min 95 % A and 5 % B, 6–45 min 95 % A and 5 % B to 100 % B (linear gradient). All these products were detected at 254 nm. Standard calibration curve of isovaleryl-CoA was generated with a series of known concentrations of the isovaleryl-CoA standard purchased from Sigma-Aldrich (Milwaukee, WI, USA). Standard calibration curves of HIBP and PIVP were generated with a series of known concentrations of the purified compounds from large scale fermentation. All experiments were carried out in triplicate and repeated at least twice. The titer was presented as mean ± SD.

1D- and 2D-NMR spectra were recorded in CDCl₃ with a Bruker AVANCE II 400 spectrometer (Bruker, Billerica, MA, USA) operated at 400 MHz for ¹H and at 100 MHz for the ¹³C nucleus. Tetramethylsilane (TMS) was used as the internal reference (δ 0.00). The high resolution mass spectra were determined in a Bruker MicroQTOF-II mass spectrometer (Bruker), equipped with an ESI source operated in positive mode at 180 °C with a capillary voltage of 4500 V. Mass accuracy was verified by calibration before and after sample introduction, using sodium formate (1 mM). Both samples and calibrant were introduced using a syringe pump at 10 µL min⁻¹. Electron impact mass spectra (EI-MS) were obtained at 70 eV by GC–MS on a Hewlett–Packard 5970 Series mass spectrometer interfaced (Hewlwtt-Packard, Palo Alto, CA, USA) with a Hewlett–Packard 5890 gas chromatograph fitted with a column (HP-5MS, 15 m × 0.25 mm i.d., temperature from 200–290 °C, 10 °C/min). HPLC was performed in a Shimadzu chromatograph with a diode array UV-Vis detector (Shimadzu, Kyoto, Japan). Fourier transform infrared spectra were acquired on a FTIR Nicolet 510P spectrometer (Thermo Scientific, Waltham, MA, USA). Spectra over a range of 4000–500 cm⁻¹ with a resolution of 2 cm⁻¹ (50 scans) were recorded using KBr pellets.