1260 infinity hplc system

Metalloproteomic culture cell pellets were resuspended in 200 μL 20 mM Tris-HCl pH 8.0 and sonicated using a Bioruptor system (Diagenode) for 25 cycles (30 s on, 30 s off). Sonicated cultures were then centrifuged at 4 °C for 15 min at 18,000 × g to remove insoluble material. The supernatant was harvested and 2 mg total protein was fractionated via AEXchromatography using a Bio IEX 3 μm column (Agilent) on an Infinity 1260 HPLC system (Agilent). AEX fractions were collected in 1-min intervals, corresponding to 400 μL fractions. AEX fractions were then independently separated via SEC using a Bio SEC-3 column (Agilent) on an Infinity 1260 HPLC system (Agilent), directly hyphenated to an ICP-MS 7500cx (Agilent) to determine metal content. Detection of metals (recorded in counts per second) were normalized to an internal standard (antinomy [Sb]) and metal concentration (in ppb) was interpolated from a calibration curve of known metal concentrations. Determination of protein-metal interactions (denoted by number of peaks with increased metal abundance) was conducted using baseline-corrected area under curve analysis (GraphPad Prism 8.0). Metalloproteomic maps were generated using MATLAB 2020a.

To analyze the phenolic content of the different green tea extracts, high-performance liquid chromatography/time-of-flight mass spectrometry (HPLC-TOF/MS) analysis was used. Agilent Technologies 1260 Infinity HPLC System was coupled to a 6210 time-of-flight (TOF) LC/MS detector and ZORBAX SB-C18 (4,6 × 100 mm, 3.5 μm) column. The mobile phases A and B were ultrapure water solution with 0.1% formic acid and acetonitrile, respectively. Flow rate was 0.6 ml/min and column temperature was 35°C. The green tea extracts (200 ppm) and stock solutions of 23 standard phenolic compounds (2.5 ppm) were prepared in methanol at room temperature. The samples were filtered passing through a PTFE (0.45 μm) filter by an injector to remove particulates. The injection volume was 10 ml and the solvent program was as follows: 0 min 10% B; 0-1 min 10% B; 1–20 min 50% B; 20–23 min 80% B; 23–25 min 10% B; 25–30 min 10% B. The ionization mode of MS-TOF instrument was ES negative with gas temperature of 325°C, gas flow of 10.0 l/min, and nebulizer of 40 (psi). The phenolic content of green tea extracts was determined by comparing retention times and m/z values of green tea extracts and standard phenolic compounds.

Amino acidphenylisothiocyanate/PITC derivatization was performed based on a previous method (Kameya et al. 2007 (link); Ma’ruf et al. 2021 (link)) with certain modifications; for example, we used a dry bath instead of a vacuum concentrator. 10 µL of reaction product was added to 10 µL of 50 mM L-alanine in a microtube and then dried at 80 °C in a dry bath for 30 min. The sample was dissolved with 20 µL of ethanol: milliQ: triethylamine (2: 2: 1) and dried for 20 min at 80 °C. The precipitate was then dissolved with 20 µL of ethanol: milliQ: triethylamine: PITC (7: 1: 1: 1). This was followed by incubation at room temperature for 20 min and a drying process for 25 min at 80 °C. The sample was dissolved with 500 µL of methanol. HPLC analysis was performed by Agilent Technologies 1260 Infinity HPLC system using ZORBAX Eclipse XDB-C18 column (4.6 × 250 mm, 5 µm). Two types of buffers–A buffer (15 mM phosphate buffer pH 7) and B buffer (methanol LC grade)–were used for the elution process. The samples were run for 36 min, with B buffer gradient from 0 to 75% for the first 30 min. The buffer concentration was then reduced to 0% for the remaining 6 min.