C18 analytical column

The PDA detector combined with an Agilent 1100 liquid chromatography system was used to perform a qualitative and quantitative ursolic acid content analysis of the extracts. In the analysis, Thermo C18 analytical column (250 × 4.6 mm, 5 µm particle diameter) as the stationary phase, acetonitrile: methanol (80:20) system was preferred as the mobile phase. The mobile phase passed through the system isocritically at a flow rate of 1 mL/min and the total analysis time for one sample was set for 15 min. The detection wavelength was 210 nm. Solutions were prepared at concentrations ranging from 7.8–2000 µg/mL to obtain a calibration curve for ursolic acid (Sigma Chemical Company, St. Louis, MO, USA) purchased as standard. The regression equation was obtained as y = 8.9087x + 143.24 and the calibration plot showed good linearity with a correlation coefficient of 0.9998. Limits of detection (LOD) and limit of quantification (LOQ) of the ursolic acid standard were found 0.045 and 0.136 mg/mL respectively. Extracts were prepared at a concentration of 10 mg/mL, filtered, and then injected into the system. Each analysis was repeated three times [62 (link)].

Labeled samples were separated through online reversed-phase chromatography using Easy nLC1000 system (Thermo Fisher Scientific, United States). The peptides were autoloaded into a C18 trap column (2 cm × 100 μm, 5 μm; Thermo Fisher Scientific, United States), and subsequently eluted into a C18 analytical column (75 μm × 100 mm, 3 μm; Thermo Fisher Scientific, United States) for gradient elution at a flow rate of 250 nL/min for 120 min. LC-MS/MS was conducted using a Q-Exactive (Thermo Fisher Scientific, United States) mass spectrometer. The procedure was performed in positive ion mode with MS1 survey scan (m/z: 300–1800) at a resolution of 70,000, followed by 10 higher-energy collisional dissociation (HCD) type MS2 scans with a resolution of 17,500.

LC‒MS/MS data acquisition was carried out on Orbitrap Exploris 480 mass spectrometer coupled with an EASY-nLC 1200 system (both from Thermo Scientific) [43 (link)]. Peptides were picked up by an autosampler and transferred to a C18 analytical column (75 μm × 25 cm, 1.9 μm particle size, 100 Å pore size, Thermo) for separation. Mobile phase A (0.1% formic acid) and mobile phase B (80% ACN, 0.1% formic acid) were used for the 60 min gradient separation procedure. A constant flow rate of 300 nL/min was used. For DDA mode analysis, each cycle consisted of the acquisition of one full scan mass spectrum (R = 60 K, AGC = 300%, max IT = 20 ms, scan range = 350–1500 m/z) followed by 20 MS/MS events (R = 15 K, AGC = 100%, max IT = auto, cycle time = 2 s). The HCD collision energy was set to 30. The isolation window for precursor selection was set to 1.6 Da. The former target ion exclusion was set to 35 s.

Peptide mixtures were separated using nano-LC system (UltiMate 3000 RSLC, Thermo Scientific, Waltham, MA, USA) and reversed-phase chromatography. Peptides samples were trapped on trap column (Thermo Scientific, USA) and peptide separation was conducted on C18 analytical column (Thermo Scientific, USA). Peptides were eluted using a 150-min gradient using buffer A (5% dimethyl sulfoxide with 0.1% formic acid) and buffer B (5% dimethyl sulfoxide with 0.1% formic acid in 80% acetonitrile) and [18 (link)] at 50 °C (buffer B from 5 to 40% over 150 min, from 40 to 95% over 2 min, and kept at 95% for 23 min, then from 95 to 5% over 10 min and kept at 5% for 15 min) at 250 nL/min. The mass spectrometry analysis was performed using Q Exactive Plus mass spectrometer (Thermo Scientific). Data-dependent acquisition was performed with an automatic switch between a MS1 (m/z 350-1800) and 20 MS2 scans. MS1 spectra were measured with an AGC of 3e6, a resolution of 70,000 and an injection time of 100 ms. MS/MS spectra were triggered at an AGC of 1e5, a resolution of 17,500, and a normalized collision energy of 27. Repeated peptides were excluded for 20 s. Three technical replicates were applied for each sample.

Peptides were re-dissolved in solvent A (A:2%ACN + 0.5% acetic acid) and separated through the nanoflow CHIP-LC system C. The peptides were auto-loaded into the Dionex Acclaim Pepmap100 C18 trap column (2 cm × 100 μm, 5 μm; Thermo Fisher Scientific, United States), and subsequently eluted into a C18 analytical column (75 μm × 25 cm, 2 μm; Thermo Fisher Scientific, United States) for gradient elution from 4% solvent B (B: 98%ACN + 0.5% acetic acid) to 100% solvent B in 150 min. LC-MS/MS was performed using the TripleTOF mass spectrometer (AB Sciex, United States).