Dionex ultimate 3000 hplc system

MS analysis of phospholipids was performed on an Orbitrap mass spectrometer (Thermo Fisher). Phospholipids were separated on a normal phase column (Luna 3 µm Silica (2) 100 Å, 150 × 2.0 mm, Phenomenex, Torrance, CA, USA) at a flow rate of 0.2 mL min⁻¹ on a Dionex Ultimate 3000 HPLC system (Dionex, Idstein, Germany). The column was maintained at 35 °C. Analysis was performed using gradient solvents (A and B) containing 10 mM ammonium acetate. Solvent A contained propanol:hexane:water (285:215:5, vol/vol/vol) and solvent B contained propanol:hexane:water (285:215:40, vol/vol/vol). All solvents were LC–MS grade. The column was eluted for 0–23 min with a linear gradient of 10–32% B; 23–32 min using a linear gradient of 32–65% B; 32–35 min with a linear gradient of 65–100% B; 35–62 min held at 100% B; 62–64 min with a linear gradient from 100% to 10% B followed by and equilibration from 64 to 80 min at 10% B. The instrument was operated with the electrospray ionization probe in negative polarity mode. Data was analyzed using Quan Browser of xcalibur software (Thermo Fisher).

100 µg protein lysates from each sample were reduced with 10 mM DTT for 45 min at 37 °C and alkylated with 55 mM chloro-acetamide for 30 min at room temperature in the dark. Samples were diluted with 5 volumes of 40 mM Tris–HCl, pH 7.6 and hydrolyzed with trypsin (Promega, Mannheim, Germany) in a 1:50 (w/w) enzyme-to-substrate ratio during overnight incubation at 37 °C in a thermoshaker at 700 rpm. Samples were acidified with formic acid (FA) to a concentration of 0.5% (v/v). Samples were desalted using self-packed stage-tips [10 discs, Ø 1.5 mm, C18 material, 3 M Empore™ Octadecyl C18, Saint Paul, MN, USA; wash solvent: 0.1% formamide (FA); elution solvent: 60% acetonitrile (ACN) in 0.1% FA]. 100 µg of protein hydrolysate were dissolved in 50 mM HEPES, pH 8.5 and mixed for 10 min at 20 °C. Tandem Mass Tag (TMT) reagents (Thermo Fisher Scientific) were added to each protein hydrolysate to a final concentration of 11.6 mM and incubated at 400 rpm on a thermomixer for 1 h at 20 °C. Reactions were stopped with 0.4% hydroxylamine (v/v). Labelled peptide solutions were pooled and desalted on Sep-Pak tC18 RP extraction cartridges (Waters Corp., Finglas, Ireland; wash solvent: 0.1% FA; elution solvent: 60% ACN in 0.1% FA). TMT-labelled samples were fractionated using a Dionex Ultimate 3000 HPLC System (Dionex Corporation, Idstein, Germany) and collected in 32 fractions.

FPE extracts and standard compounds including Puerarin (98%, P1886, Tokyo Chemical Industry, Tokyo, Japan), Apigenin (98%, A1514, Tokyo Chemical Industry), Genistein (98%, G6776, Sigma-Aldrich, Saint Louis, MO, USA) and Rutin (95%, Zhejiang Medicines & Health Products Imp. & Exp Co, Hangzhou, China) were analyzed using Thermo Scientific™ Dionex™ UltiMate™ 3000 HPLC System with UV detector (Dionex, Sunnyvale, CA, USA). Gradient elution was carried out on Inno-C18 (250 mm × 4.6 mm, 5 μm) with the mobile phase consisted of CH3CN and 0.3% HCOOH aqueous solvent. The flow rate was 0.8 mL/min. Column temperature was held constant at 45 °C. The UV detection wavelength was 280 nm, 366 nm. Standard and sample (10 μL) were subjected to HPLC analysis.

All reagents were purchased from Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA) and were of the highest purity commercially available. UV spectra were obtained with a Varian Cary 300 Bio UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). HPLC was performed on a Dionex Ultimate 3000 HPLC system (Thermo Electron, Madison, WI, USA) equipped with a diode array detector using a Macherey-Nagel C18 reverse-phase column (Macherey-Nagel, Bethlehem, PA, USA). Radiolabeled samples were counted in a Beckman LS6500 scintillation counter (Beckman, Indianapolis, IN, USA).