Nexera uhplc

PGE2, PGE2-d9, PGD2, 15-keto-PGF2α, PGA2, PGB2, and PGJ2 analytical standards were purchased from Cayman Chemical (Ann Arbor, MI). All solvents used for LC-MS were HPLC Plus grade from Sigma Aldrich Corp. (St. Louis, MO).
Cell culture supernatants along with relevant standards were undergone extraction and samples were analyzed by LC-MS using a Shimadzu Nexera UHPLC (Shimadzu North America, Columbia, MD) interfaced to a Q-Exactive Plus ion trap mass spectrometer with a HESI source (ThermoFisher Scientific, San Jose, CA). Chromatographic separations were achieved by employing a 2.1 x 100 mm, 1.8 μm, Acquity HSS T3 column (Waters, Milford, MA) with gradient elution at 0.45 mL/min [20 (link)]. PGE2 was quantified against PGE2-d9 internal standard. The standard curve was generated using a constant concentration of PGE2-d9 (50 pg on column) and variable concentrations of PGE2 (2 pg to 10 ng on column). Peak areas for LC-MS quantification of PGE2 and PGE2-d9 were calculated using Xcalibur QuanBrowser software (ThermoFisher Scientific, San Jose, CA). The coefficient of determination for the standard curve (R2) was 0.9996.

Crude peptides obtained from either plant extracts or chemical synthesis were purified using a series of Phenomenex C18 columns on RP-HPLC. Gradients of 1%/min of 0–80% solvent B (90% MeCN in 0.045% TFA in H₂O) and solvent A (aqueous 0.05% TFA in H₂O) were used and the eluent was monitored at 215 and 280 nm. Peptide purity was examined on a Nexera UHPLC (Shimadzu) with a flowrate of 0.4 mL/min on a 0.8 mL/min Agilent column using a 2% gradient of 0–50% solvent B and masses were determined by electrospray mass spectrometry. All peptides used in assays had >95% purity. Folding trials were carried out using 1 mg/mL aliquots of peptide in various folding buffers of different pHs, as well as different concentrations of ammonium bicarbonate and dimethyl sulfoxide (DMSO). The optimal conditions for peptide folding were as follows: SFTI-1 grafted peptides were folded in two steps: cyclization with 0.1 M ammonium bicarbonate (pH 8.0)/0.1 M TCEP, then oxidation in 0.1 M ammonium bicarbonate (pH 8.0); MCoTI-II grafted peptides were folded in a one-pot oxidation solution of 0.1 M ammonium bicarbonate (pH 8.5) except for MCo-SST-01, MCo-SST-02, MCoAA-01 and MCoAA-02, which required the addition of 10% DMSO. Examples of LC-MS traces of folded MCoAA-01 and MCoAA-02 with the above conditions are provided in Supplementary Fig. S6.

The compound at 20 μM (2% final DMSO concentration) was incubated with CD-1 mouse (Innovative Research), SD rat (Innovative research), or human plasma (George King BioMed) at 37°C for up to three hours. Aliquots were taken out at different time points and quenched in acetonitrile containing diclofenac (internal standard). The samples were then centrifuged and the supernatant was analyzed by UHPLC using a Kinetex 2.6 μm C18 (Phenomenex) column and a Shimadzu Nexera UHPLC. Verapamil was used as a positive control.

To identify the bioactive molecule(s) in the aqueous extract derived from Enterobacter sp. 84.3, ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) was carried out. The analyses were performed in a modular Nexera UHPLC (Shimadzu, Kyoto, Japan) system with LC-30AD pump, SIL-30AC autosampler, CTO-30A column oven, and SPD-M20A DAD detector (spectra were recorded from 200 to 800 nm). The LC-system was connected to a QTOF-HRMS mass spectrometer (Bruker, Billerica, MA, USA) fitted with an ESI source (negative mode). Negative ion mass spectra were recorded in the range of m/z 50–1.500 at a scan speed of 1.5 kDa/s at a voltage of −5 kV and capillary temperature of 280 °C. Nitrogen was used as the gas source. The components were separated using a Shimpack XR II series C18 (120 mm × 3.6 mm × 2.1 μm). Samples were solubilized in methanol (MeOH) MS-LC grade solvent (3 mg/mL), and the injection volume was 3 μL for the samples. The mobile phases used were 0.1% (v/v) formic acid in water (phase A) and acetonitrile (phase B) at a flow rate of 0.3 mL/min. The gradient program was as follows: 3% B (0.01 min), 3–15% B (10 min), 15–45% B (12 min), 45–100% B (4.5 min), 100% B (2.5 min), and 100–0% B (4 min).

Lyophilized turgencin B (0.5 mg) was partially reduced in 210 µL of 0.17 M citrate buffer (32 mg/mL citric acid, 50 mg/mL trisodium citrate dihydrate) (pH 3.0). Peptide sample was then combined 1:1 with cold (4 °C) 10 mM tris(2-carboxyethyl)phosphine (11.4 mg/mL) solution in 0.17 M citrate buffer (pH 3.0). One sample of turgencin B was incubated at 40 °C, a second sample at room temperature, and a third sample at 4 °C. Aliquots of 50 µL were taken out at different timepoints (10, 20, 30, 45, 60, 90, 120 and 240 min) from all three samples. Upon sampling, each reaction solution was immediately added to 0.6 mg of pre-prepared N-ethylmaleimide (NEM) and incubated for 15 min at 37 °C before acidification through addition of 300 µL of 1% FA. After this step, salt was removed from all samples using C18 ZipTips (Millipore), and eluted in 10 µL of 80% MeCN with 1% FA. Following this, 100 µL of 0.5 M ammonium bicarbonate (pH 8.0) was added. Samples were fully reduced by adding 35 µL of 100 mM dithiothreitol, deoxygenated with N₂, and incubated at 60 °C for 30 min. Afterwards, they were alkylated by adding 35 µL of 250 mM iodoacetamide for 15 min at room temperature. The reduced and alkylated samples were analyzed on a Nexera UHPLC (Shimadzu) coupled to a TripleToF 5600 MS (AB Sciex).

Analysis of cyantraniliprole and IN-J9Z38 was performed on a LCMS-8040 (Shimadzu, Japan) coupled with Nexera UHPLC (Shimadzu, Japan). Positive mode in electro-spray ionization (ESI) was used for the MS/MS detection with the following MS parameters: the capillary voltage was 4.0 kV, and nitrogen was the nebulizing gas (3.0 L/min) and drying gas 15.0 L/min). The desolvation line temperature was 250 °C and the heat-block temperature was 400 °C. Chromatographic separation for UHPLC was carried out on a Kinetex C18 column (100 mm × 2.1 mm, 2.6 μm, Phenomenex, CA, USA) at a 40 °C column temperature. The mobile phase consisted of deionized water (A) and methanol (B), both containing 0.1% formic acid and 5 mM ammonium formate. The flow rate of the mobile phase was 0.2 mL/min and the gradient was programmed as follows: initially, 5% of the organic solvent mobile phase (B) was kept constant for 1 min followed by increasing it to 95% (B) linearly over 2.5 min and held for 3.5 min. Finally, the ratio of B was restored to 5% over 0.5 min and maintained for 2.5 min. The total analytical running time was 10 min and the injection volume was 5 μL. Highly selective MS/MS detection was achieved by multiple reaction monitoring (MRM). The MRM transition pair of quantitation and identification was optimized by direct injection of standard solution (1 μg/mL) without an analytical column.