Lcms 2010 ev system

The analysis was performed on a Shimadzu (Japan) LC-MS 2010 EV system equipped with an electrospray ionization (ESI) interface. The liquid chromatographic separation was achieved on CAPCELL PAK C₁₈ column (150 mm × 4.5 mm, 5 μm), which was protected by a guard column (4 mm × 2 mm, 5 μm). The mobile phase, consisting of acetonitrile (solvent A) and 0.05% glacial acetic acid water (Solvent B), was delivered at a flow rate of 0.8 mL/min with 25% of the eluent split into the inlet of mass spectrometer. The gradient program was shown as follows: 0-1 min, 13–25% A; 1–3 min, 25% A; 3–5 min, 25–80% A; 5–7 min, 80% A; 7-8 min, 80–13% A; 8–10 min, 13% A. The column and autosampler tray temperature were maintained constantly at 35°C and 4°C, respectively. The inject volume was 10 μL and the run time was 10 min.
The analytes and IS were ionized by ESI source in positive ion mode under the following source conditions: nebulizing gas 1.5 L/min, curved desolvation line (CDL) temperature 250°C, heat block temperature 250°C, detector voltage 1.95 kV, and the other parameters were fixed as tuning file. Selected ion monitoring (SIM) mode was applied for the quantification of [M + H]⁺ at m/z 335.05 for strychnine, [M + H]⁺ at m/z 395.10 for brucine, and [M + H]⁺ at m/z 748.45 for IS, respectively.

Peptide purification was accomplished using a Shimadzu LC-8A preparative HPLC system (Shimadzu, Kyoto, Japan), equipped with a SPD-M10AV UV-Vis detector. A linear gradient of H₂O 0.1% TFA (eluent A) and acetonitrile 0.1% TFA (eluent B), from 5 to 70% B over 50 min at a flow rate of 22 mL/min, eluted a Reverse Phase Vydac C18 column (250 cm × 22 mm; 10 μm).
Peptide purity and identity were assessed by RP-HPLC-MS analysis (Supplementary Fig. 10–12), using a Shimadzu LC-10ADvp equipped with an SPDM10Avp diode-array detector. ESI-MS spectra were recorded on a Shimadzu LC-MS-2010EV system with ESI interface and a quadrupole mass analyzer. A Vydac C18 column (150 mm × 4.6 mm, 5 μm) was used in the LC-MS analyses, eluted with a linear gradient of H₂O 0.1% TFA (eluent A) and acetonitrile 0.1% TFA (eluent B), from 5 to 70% B over 60 min at a flowrate of 0.5 mL/min.

One-dimensional proton nuclear magnetic resonance spectra of CDCl₃ were recorded on a JEOL JNM‐ECA‐600 (600 MHz); residual CHCl₃ (δ = 7.26 ppm) was used as an internal reference. High-performance liquid chromatography (HPLC) was performed using a Shimadzu Prominence liquid chromatography system with an SCL-10Avp system controller, an SPD-M20A photodiode array detector (300–800 nm), two LC-20AD pumps, a DGU-20A₃ degasser, and a CTO-20AC column oven. Liquid chromatography–mass spectrometry (LC–MS) was performed using a Shimadzu LCMS-2010EV system, which was based on the atmospheric pressure chemical ionization mode (Hirose et al. 2022 (link)). For all LC(-MS) analyses, the column oven was set to 30 °C.
Flash column chromatography (FCC) and reversed-phase HPLC were performed using silica gels (Merck Kieselgel 60, 0.040–0.063 mm or Wakogel C-300) and a packed octadecylated silica gel column (Cosmosil 5C₁₈-AR-II, Nacalai Tesque), respectively. The sample was dissolved in an HPLC eluent. The solution was filtered using a Cosmonice filter (0.45 μm pore size, Nacalai Tesque), and the filtrate was subjected to HPLC. Methanol and distilled water for HPLC solvents were purchased as HPLC grade from Nacalai Tesque. Acetone for HPLC grade was purchased from FUJIFILM Wako Pure Chemical.

PCR reagents, plasmid purification, restriction enzymes, and T4 DNA ligase were used as recommended by New England Biolabs (NEB) unless otherwise noted. Vectors used in this study included the bacterial expression vectors pCWori (43 (link)) and pET28a (Novagen). E. coli cell strains used in this study included Top10 (Invitrogen), DH5α, and BL21(DE3) (NEB). All reagents were used as received from commercial sources. Loganin (Cayman Chemical) and loganic acid (Arctom Chemicals) were 98+% pure.
LC-MS analyses of the Camptotheca SLAS mutants were conducted using a Shimadzu LC-MS2010EV system at the Institute for Genomic Biology, University of Illinois Urbana-Champaign. High resolution LC-MS analyses of the WT Camptotheca SLASs and the SLS, SLAS common ancestor were performed by Dr Alexander Ulanov in the Metabolomics Laboratory of the Roy. J. Carver Biotechnology Center at the University of Illinois Urbana-Champaign using a Dionex Ultimate 3000 series HPLC system (Thermo Scientific) with Q-Exactive MS system (Thermo Scientific). Spectroscopic measurements were recorded with a Cary UV-Vis Bio100 dual beam spectrophotometer or Molecular Devices SpectraMax M-series plate reader as appropriate.