U3000 hplc system

The extraction and quantification of flavonoid were carried out according to previous studies with minor modification [48 (link)]. Briefly, 0.5 g of dried powder was added to 10 mL of 65% ethanol and then extracted with ultrasonic wave at 50 °C for 30 min. After centrifuged at 10,000× g for 10 min, the supernatant was collected and the absorbance was measured with an ultraviolet spectrophotometer (MAPADA, Shanghai, China) at 340 nm. The quantification of luteoloside was conducted using Dionex U3000 HPLC system (Dionex, Sunnyvale, CA, USA) with a Welch LP-C18 column (5 μm, 150 mm × 4.6 mm) at 25 °C. The flow rate was 1 mL/min and the mobile phase consisted of acetonitrile (eluent A) and water: acetic acid (999:1, v/v, eluent B). The elution program was 0–5 min, 8–10% A; 5–25 min, 10–20% A; 25–45 min, 20–30% A; 45–55 min, 30–100% A. 10 μL of the extracted sample was injected and fractions were monitored at 350 nm. Commercial luteoloside was used as the standard to identify and quantify the luteoloside contents in all samples. All results were representative of three independent experiments.

¹H, ¹³C, and 2D (correlated spectroscopy, heteronuclear single quantum coherence spectroscopy, heteronuclear multiple bond correlation) NMR measurements were performed using deuterated dimethyl sulfoxide (DMSO) and deuterated chloroform solutions using Bruker Avance-400, -600 and -800 instruments (Bruker, Billerica, MA, USA). Solvent peaks at δ_H 2.50/δ_C 39.50 and δ_H 7.26/δ_C 77.16 were applied as internal standards for deuterated DMSO and chloroform, respectively. Low-resolution electrospray ionization mass spectrometry was performed using the Agilent Technologies 6130 quadrupole mass spectrometer connected to the Agilent Technologies 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA). High-resolution ESI mass spectrometric data were obtained at the National Instrumentation Center for Environmental Management (Seoul, Korea) on a Q-TOF 5600 instrument equipped with a Dionex U-3000 HPLC system. Optical rotations were measured on a JASCO P2000 polarimeter (Jasco, Tokyo, Japan) using a 1 cm cell. Semi-preparative HPLC separations were performed using the Spectrasystem p2000 pump equipped with the Spectrasystem RI-150 refractive index detector (Thermo Scientific, Waltham, MA, USA). All solvents used were spectroscopy grade or distilled in glass prior to use.

Optical rotations were measured on a JASCO P1020 polarimeter (Jasco, Tokyo, Japan) using a 1 cm cell. UV spectra were acquired with a Hitachi U-3010 spectrophotometer (Hitachi High-Technologies, Tokyo, Japan). IR spectra were recorded on a JASCO 4200 FT-IR spectrometer (Jasco, Tokyo, Japan) using a ZnSe cell. ¹H and ¹³C NMR spectra were measured in DMSO-d₆, CDCl₃, or CD₃OD solutions on Bruker Avance –400, –500, –600, or –800 instruments (Billerica, MA, USA). High resolution FAB mass spectrometric data were obtained at the Korea Basic Science Institute (Daegu, Korea) and were acquired using a JEOL JMS 700 mass spectrometer (Jeol, Tokyo, Japan) with meta-nitrobenzyl alcohol (NBA) as the matrix. High-resolution LC-MS/MS data were obtained at the National Instrumentation Center for Environmental Management (Seoul, Korea) on a Q-TOF 5600 instrument equipped with a Dionex U-3000 HPLC system. Semi-preparative HPLC separations were performed on a Spectrasystem p2000 equipped with a Spectrasystem RI-150 refractive index detector. All solvents used were spectroscopic grade or distilled from glass prior to use.

Next, 5 mg of purified PcP was added to 1 mL of the 2.0 M trifluoroacetic acid (TFA) solution and kept in a sealed container at 121°C for 2 h for hydrolysis. The hydrolyzed sample was then dried on a nitrogen blowing instrument. After the residue was rinsed with methanol three times and dried under the protection of nitrogen, it was dissolved in ddH₂O for 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization. The sample and monosaccharide standards were analyzed on an U3000 HPLC system (Thermo Fisher, United States) equipped with a ZORBAX Eclipse XDB-C18 column, and the column temperature was set to 30°C. Chromatographic conditions were set as follows: A solvent of acetonitrile and B solvent of sodium phosphate buffer (pH = 6.8) at the ratio of 17: 83 were used to produce a 60-min isocratic elution at a flow rate of 0.8 mL/min, with an injection volume of 10 μL and detection wavelength 250 nm. The respective chromatographic peaks of monosaccharide standards were manually integrated, and sugar concentrations in the sample were quantified using an external calibration curve.

The extracted peptides were separated on a U 3000 HPLC system (Thermo Fisher Scientific). A PepMap Acclaim analytical column of 75 μm inner diameter × 50 cm length (2 μm C18 particles, Thermo Fisher Scientific) was used. The injected peptides were eluted at a flow rate of 300 nl/min with 5–40% acetonitrile gradient and subsequently ionized by on-line electrospray ionization at a potential of 2 kV, and sent into a Q Exactive tandem mass spectrometer (Thermo Fisher Scientific). In MS, the ionized peptides intact masses were measured in the Orbitrap mass analyser. In each scan, the top 10 peptides with more than a charge state of 2 + were passed on to high-energy collision cell and fragmented into y and b fragment to acquire MS/MS spectra in the orbitrap.

SCX chromatographic separations were performed on a Dionex U3000 HPLC System using a 5 μm SCX HPLC column (BioBasic, Thermo Scientific). Specifically, 160 μg of trypsin-digested HEK 293T lysate was lyophilized overnight and re-dissolved in 16 μl of SCX Solvent A (25% MeCN, 5 mM KH₂PO₄, pH 2.7), followed by loading onto the SCX Solvent A equilibrated column. After 5 min, the peptides were eluted with SCX Solvent B (25% MeCN, 5 mM KH₂PO₄, 500 mM KCl, pH 2.7) using a gradient from 0% to 50% B in 75 min followed by 50% to 100% B in 5 min and then maintained at 100% B for 10 min at a flow rate of 50 μl/min (Supplementary Fig. 11). To obtain a cationic compound-depleted fraction, the flow was collected from the 4^th to the 40^th min, lyophilized and desalted using HyperSep C18 SpinTips. Afterwards, the peptide concentration was determined by amino acid analysis before undergoing MIP- or TiO₂-based phosphopeptide enrichment.

All LC-HRMS and LC-HRMS² analyses were performed on a Thermo LTQ Orbitrap XL high-resolution ESI mass spectrometer coupled to a Thermo U3000 HPLC system. Experiments were performed with a Kinetex 5 µm, 100 mm × 2.1 mm PFP column (Phenomenex, Torrance, CA, USA), kept at 25 °C, using an elution gradient of H₂O and MeOH running and a flow rate of 200 μL/min. The gradient program was as follows: 10% MeOH for 1 min, 10−100% MeOH over 30 min, and 100% MeOH for 10 min. Mass spectra were acquired in positive ion detection mode, with resolution set to 60,000 in the range of m/z 150–2000. MS parameters were set as follows: a spray voltage of 4.80 kV, a capillary temperature of 285 °C, a sheath gas rate of 32 units N₂ (ca. 320 mL/min), and an auxiliary gas rate of 15 units N₂ (ca. 150 mL/min). Data were recorded with data-dependent acquisition (DDA) mode, in which the four most intense ions in the full-scan mass spectrum were subjected to high resolution tandem mass spectrometry (HRMS²) analysis. HRMS² scans were achieved for selected ions with collision induced dissociation (CID) fragmentation, an isolation width of 3.00 Da, a normalized collision energy of 35 units, an activation Q of 0.250 units, and an activation time of 30 ms. Mass data were analyzed using the Thermo Xcalibur software version 2.2 (Thermo Fisher Scientific Inc., Waltham, MA, USA).