Hp msd triple axis detector

GC/MS analysis was conducted on a HP GC 7890A chromatograph coupled to an anion-trap mass spectrometer HP MSD triple axis detector (Agilent Technologies, Santa Clara, CA, USA). Compounds were separated on a HP-5MS capillary column (30 m × 0.25 mm × 1.0 mm) with an injection port temperature of 250 °C and helium as carrier gas. The oven temperature started at 60 °C and was increased at 10 °C min⁻¹ to a final oven temperature of 250 °C. Mass spectra were scanned over the range 5 – 300 amu at 1 sec intervals. The retention indices and mass spectra of compound peaks were compared to reference spectra available in reference libraries in Mass Finder 4 (Joulain and Konig, 1998 ) and the National Institute of Standards and Technology (NIST) standard reference database.

In vitro assays with purified sesquiterpene synthases were conducted in Buffer D (50 mM Tris.HCl, 100 mM NaCl, pH 7.5) supplemented with MgCl₂, CaCl₂, MnCl₂, NiCl₂, CoCl₂, ZnCl₂ or KCl (10 mM) in a final reaction volume of 100 µL. Sesquiterpene synthases (10 µg) were incubated with (2E,6E)-FPP (2 µM) in a sealed glass vial for 16 hours at 21 °C. In all cases a negative control (reaction mixture without supplemented metal ions) was conducted to confirm that no extraneous metal ions were present in the system. The headspace of the reaction vessels was sampled for 10 min by a solid-phase microextraction (SPME) fiber followed by GC-MS analysis. GC-MS was conducted on an HP GC 7890A chromatograph coupled to an anion-trap mass spectrometer HP MSD triple axis detector (Agilent Technologies). Compounds were separated and mass spectra were scanned as described previously ^{[22 (link)]}. The retention indices and mass spectra of peaks were compared to reference spectra in the terpene libraries available from MassFinder 4 ^{[43 ]} and the National Institute of Standards and Technology (NIST) standard reference database.

Volatile sesquiterpenoids were extracted from E. coli and fungal culture headspace with a 100 μm polydimethylsiloxane-divinylbenzene (PDMS) Solid Phase MicroExtraction fiber (SPME, Supelco, Bellfonte, PA) inserted through aluminum foil seal for 10 min as described previously (27 (link)). Likewise, non-volatile, soluble sesquiterpenoids were extracted by SPME from 50 mL centrifuged culture media (2655 × g, 10 mins, Eppendorf 5810R swinging-bucket centrifuge) that was clarified by passage through a HT Tuffryn low protein binding 0.45 μm syringe filter (Pall, Port Washington, NY).
Extracted terpenoid products were separated and analyzed by GC-MS on an HP GC 7890A gas chromatography coupled to an anion-trap mass spectrometer equipped with an HP MSD triple axis detector (Agilent Technologies, Santa Clara, CA) and using an HP-5MS capillary column (30 m × 0.25 mm × 1.0 μm) with an injection port temperature of 250 °C and helium as a carrier gas. The oven temperature started at 60 °C and was increased at 6 °C min⁻¹ to a final temperature of 250 °C with a 38 min cycle time. Terpenoids were identified by comparing mass spectra and retention indices to MassFinder’s (Software v.4) terpene library (28 ) and the NIST chemical database as described previously (29 ).