Q exactive plus hybrid quadrupole orbitrap mass

Samples were processed for mass spectroscopy with Thermo Scientific Q Exactive Plus Hybrid Quadrupole Orbitrap mass spectrometer as follows. For reduction, TRX-f1 (TRX-f1 red) was incubated with 500 µM DTT for 30 min at room temperature followed by a desalting step using PD 10 column and 30 mM Tris-HCl, pH 7.9, as elution buffer. Similar to the protocol of Shibata [18 (link)] using a ligand/protein ratio of ~12, TRX-f1red was incubated with 10-fold molar excess of 12-OPDA at 35 °C for 10 min. As a control, TRX-f1red was incubated with the same volume of methanol. The end concentration of methanol in protein samples was below 1% if not stated otherwise. Then, 25 μL of the samples (15 μg) were desalted, eluted in 30 μL of 80% acetonitrile and 0.1% formic acid and diluted with 470 μL 50% methanol and 0.1% formic acid. Q Exactive Plus intact protein mode was activated and sample injected at 20 μL min⁻¹ (ESI Source). Myoglobin (5 pmol µL⁻¹) was measured as a standard and the resolution was 140,000. Results derive from measuring the same samples at different modes, these were in-source 0, 20, 30 and 40. Deconvoluted masses were calculated as neutral masses.

First, 1 mg of P. taiwanensis Hayata crude extract was dissolved in 1 mL of ethanol (ACS Grade). We then took the sample solution and diluted it 100 times with methanol (ACS Grade). The solution was filtered through a 0.22 μm filter to inject 1.0 μL for UHPLC-MS/MS analysis. Q Exactive Plus Hybrid Quadrupole Orbitrap Mass (Thermo Fisher Scientific Inc., USA) was used for qualitative and quantitative analyses to characterize the chemical composition of P. taiwanensis Hayata. It was subsequently analyzed on a Accucore TM Polar Premium column (150 × 2.1 mm, 2.6 µm) (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 40 °C. The mobile phase was water (A) and 100% methanol (B) at a flow rate of 0.3 μL/min. The gradient was 0–1 min, 80% A; 1–25 min, 80–0% A; 25–30 min, 0% A; 30–30.1 min, 0–80% A; 30.1–35 min, 80% A, with an injection volume of 10 μL. Spray voltages were 3.5 kV in positive mode and −3.2 kV in negative mode, capillary temperature was 360 °C, source heater temperature was 350 °C, resolving power was 15,000, scan range was m/z 100–1000, higher-energy collisional dissociation (HCD) was 20, 30, 40, and 50 eV, and the process software was Xcalibur (version 2.2).