Prominence high performance liquid chromatography system

SEC–SAXS measurements were conducted with a laboratory-based SEC–SAXS system (La-SSS) (Inoue et al., 2019 ▸ ), which is made up of a NANOPIX combined with a Prominence high-performance liquid chromatography system (SHIMADZU, Japan). A Superdex 200 Increase 10/300 GL for BSA, Cat, βB2-cry and OVA, a Superose 6 Increase 10/300 GL for AF, and a Superdex 75 Increase 10/300 GL for Lyz and RNaseA were utilized as the SEC column. All measurements were performed at a flow rate of 0.02 ml min⁻¹ at 298 K.

All chemicals used were of the analytical reagent grade and used as received. All solvents were purchased from Daejung Chemicals (Siheung, Korea), and the standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Atlas green and white spear samples were stored at −70°C and freeze-dried for 48 h. Each sample was pulverized using a mortar and pestle. The powdered sample (0.1 g) was extracted with 1 mL of 70% methanol at 30°C for 24 h. The supernatant was collected by centrifugation at 14,000 rpm at 25°C for 10 min. After filtration using a 0.22-μm syringe filter (Woongki Science, Seoul, Korea), the filtrate was used for analysis. The sample was separated on a C18 column (250 mm × 4.6 mm, particle size 5 μm; Shimadzu, Kyoto, Japan) using a Prominence high-performance liquid chromatography (HPLC) system (Shimadzu, Kyoto, Japan) equipped with a diode array UV-vis detector. Solvent A was water (formic acid 0.1%) and solvent B was acetonitrile (formic acid 0.1%). The flow rate was 0.7 mL/min, the injection volume was 10 μL, and the column temperature was maintained at 40°C. The analytical wavelengths were 205 and 330 nm. Initially, the concentration of solvent B was 12%, which was then increased to 30% and 80% at 20 and 50 min, respectively.

MS analysis of the irradiated rose bengal/2-thioHis and rose bengal/EGT samples were completed to determine what oxidized species were present. MS analysis of oxidized 2-thioHis and EGT samples with rose bengal in D₂O was completed within 30–45 min of irradiating the samples to avoid significant decomposition of any oxidation products. For these analyses, an Applied Biosystems QTrap 4000 hybrid triple-quadrupole/linear ion trap LCMS (SciEx, Framingham, MA, USA) was used. Positive electrospray ionization (ESI) was used as the ionization source. Samples were diluted with deionized water and directly infused via a Harvard Apparatus model 22 syringe pump (Harvard Apparatus, Holliston, MA, USA) at 5 µL/min into an isocratic (50% water and 50% acetonitrile with 0.1% formic acid) mobile phase flow from a Shimadzu Prominence high-performance liquid chromatography (HPLC) system (Shimadzu Scientific Instruments, Columbia, MD, USA). Mobile phase flow was maintained at 100 µL/min. Source temperature was maintained at 400 °C. Nitrogen was used for the sheath gas, auxiliary gas, and curtain gas. Sheath gas (GS1) flow was set at 40, auxiliary gas flow (GS2) at 50, curtain gas flow (CUR) at 30, and the declustering potential (DP) was set to 50. The mass spectrometer was operated in single quadrupole mode, scanning from m/z 100 to 1000.