Waters alliance 2695

The l-dopa content was determined as described previously [3 (link)]. Briefly, 100 µL of the supernatant were diluted with 900 µL of 50% aqueous methanol. After filtration through a 0.45-µm membrane filter, the samples were analyzed by gradient elution on a C18 column (Prontosil 120-5-C18-SH 5.0 μm (250 × 4.6 mm), Bischoff, Leonberg, Germany) with an HPLC (Waters 2695 Alliance, Waters Inc., Milford, MA, USA). Solvent A was 0.3% formic acid in water (v/v) and solvent B was 0.3% formic acid acetonitrile. The following gradient was used for solvent B: 2% (0–9 min), 2–80% (9–10 min), 80% (10–14 min), 80–2% (14–16 min), and 2% (16–20 min). An injection volume of 5 µL and a flow rate of 0.8 mL/min were used. The peak of l-dopa was detected at 280 nm by a Waters 996 photodiode array detector (Waters Inc.).

The main components of MFAE were analyzed and semi-quantitated by high-performance liquid chromatography analysis (HPLC) method with the reference standard of compounds 1–4, which were isolated previously from the extracts of MFAE by author; structures were elucidated by comparison of spectral data (¹H NMR and ¹³C NMR) with the literature data. The purity of compounds 1–4 was above 98% by LC analysis based on a peak area normalization method. The semi-quantitated HPLC analysis method was done according to reference [19 (link),20 (link)].
The HPLC System Waters 2695 Alliance combined with Waters 2998 PDA detector was used for qualitative analysis of MFAE. The SHIMADZU C18 LC column (150 × 4.6 mm, 5 μm), with a column temperature of 30°C, was applied. The gradient elution process with the mobile phase (A, 1% formic acid in water; B, acetonitrile) is as follows: 0–5 min with 15–21% of mobile phase B, 5–22 min with 21–50% of mobile phase B, 22–32 min with 50–80% of mobile phase B, 32–40 min with 80–98% of mobile phase B, 40–45 min with 98% of mobile phase B, 45–47 min with 98 to 15% of mobile phase B, 47–55 min with 15% of mobile phase B. The flow rate was 1 ml/min.

The molecular weight distributions of rice protein and MRPs were estimated by HPLC (Waters 2695 Alliance, Waters Inc., USA), Waters liquid Chromatography system equipped with a Ultrahydrogel™ 1,000 column (7.8 × 300 mm, Waters, USA). The column was maintained at 30°C and operated at a flow rate of 0.5 ml/min with 50 mM carbonate buffer (pH 9.5) and the effluent was monitored at 280 nm using a refractive index detector (2414 Waters Inc., USA). The samples were dissolved in the mobile phase to reach a final concentration of 0.1% (w/v protein) and filtrated with 0.45 μm filter membrane.

Endogenous melatonin was extracted from the Arabidopsis plants using the buffer solution (acetone:methanol:water = 89:10:1) according to the method described by Pape and Lüning [52 (link)]. After protein precipitation and centrifugation, the content of melatonin was determined using melatonin enzyme-linked immunosorbent assay (ELISA) kit (EK-DSM; Buhlmann Laboratories AG, Schonenbuch, Switzerland) as recently reported by Shi et al. [16 (link)]. Additionally, the content of polyamines, including Put, Spd, and Spm, was quantified following the method described by Imai et al. [53 (link)]. Approximately 500 mg of Arabidopsis plants were homogenized in 5 mL of 5% (w/v) perchloric acid (PCA). After centrifugation at 12,000× g for 10 min, the supernatant was subjected to successive treatments including hydrolysis, dryness, and dansylation. Finally, the extracted polyamines were determined by high performance liquid chromatography (Waters 2695 Alliance; Waters, Milford, MA, USA) with a reverse phase C18 column (250 mm × 4.6 mm, 5 μm particle size). A 1 μL sample was applied and eluted with methanol into the chromatography using methanol-water (70:30, v/v) as the mobile phase at a flow rate of 0.8 mL/min, and monitored with a Waters™ 996 photodiode array detector at 265 nm.

Separation of the antibodies using size-exclusion chromatography was accomplished using a Waters Alliance 2695 (Waters, Milford, MA, USA) connected to a BioSuite High Resolution SEC Column (7.5 mm × 300 mm, 10 µm particle size, Waters, Milford, MA, USA). The separation was performed using an isocratic elution with PBS, pH 7.0, at a flow rate of 1 mL/min. The effluent was detected with a UV/Vis detector 2469 at 280 nm.
The intact masses of the antibodies were determined using reverse-phase chromatography separation. The separation for intact mass was accomplished using a Waters ACQUITY I class UPLC system (Waters, Milford, MA, USA) connected to a Thermo MabPac^TM RP column (2.1 mm × 50 mm, 4 µm particle size, Thermo Fisher Scientific). The separation was performed with eluent A, consisting of 0.1% formic acid in water, and eluent B, consisting of 0.1% formic acid in 100% acetonitrile, at a flow rate of 0.2 mL/min. The gradient was fixed for 2 min at 20% eluent B and linearly increased for 8 min from 20% to 50% eluent B. The effluent was analyzed with a Waters Synapt G2-Si HDMS system. A quantity of 50 μL (0.2 mg/mL) of antibodies was mixed with 1.5 μL (500,000 units/mL) of PNGaseF (New England BioLabs), then reacted overnight at 37 °C. Native and PNGaseF-treated antibodies were injected with 5 μL of the samples.

The content of ferulic acid (FA) in WEAX was measured with a modified method by Wang et al. [22 (link)]. WEAX (50 mg) was dissolved in 3 mL of 4 M sodium hydroxide and saponified under a nitrogen atmosphere for 18 h at RT with continuous mixing on a Bio RS-24 Mini-Rotator (SIA Biosan, Riga, Latvia). Then, the supernatant was separated by centrifugation (3000× g, 10 min, RT) and acidified to pH 2 using 6 M HCl. The FA was extracted three times with 3 mL ethyl acetate. The combined extractions were dried under nitrogen followed by dissolution in 500 μL of H₂O/MeOH solution at a 1:1 ratio. Twenty microliters of sample was injected for HPLC (Waters Alliance 2695, Waters Corporation, Milford, CT, USA) with a Capcell Pak C18 UG120 column (250 × 1.5 mm, 5 μm). The solvent system was composed of solvent A—0.1% (v/v) trifluoracetic acid (TFA) in water and solvent B—0.1% (v/v) TFA in methanol. The flow rate was 0.8 mL/min, and detection was performed on the absorbance at 320 nm. Elution was started isocratically at 35% solvent B, and then a linear gradient from 35% to 95% solvent B for 25 min was applied.