Empower

Tryptic peptide mapping with tandem mass spectrometry (MS/MS) was used to identify and quantify oxidation. Reference and oxidized samples were denatured, reduced, alkylated, and digested then separated in reversed phase liquid chromatography on a UPLC BEH C18 2.1 × 100 mm column (Waters, Milford, MA) with an Acquity UPLC H-Class system (Waters) and detected by a Q Exactive Plus mass spectrometer (Thermo Scientific, San Jose, CA). MS and MS/MS spectra were analyzed by Xcalibur (Thermo Scientific) to identify and quantify oxidation levels of tryptic peptides.
Size exclusion chromatography (SEC) was used to identify and quantify soluble aggregates from AAPH treatment. Reference and oxidized samples were separated on a TSKgel G3000SWxl 7.8 mm × 30 cm column (TOSOH Biosciences, King of Prussia, PA) on an Acquity UPLC H-Class system (Waters) with photodiode array detection at 220 nm. Absorbance traces were analyzed by Empower (Waters) to identify and quantify soluble aggregates.

Hydrophobic and hydrophilic characteristics of the peptide components of Transferon were analyzed by reversed-phase UHPLC (RP-UHPLC). All samples were filtered through a 0.10 µm polyvinylidene fluoride (PVDF) membrane (Merck Millipore Co., Darmstadt, Germany) before being injected into the UPLC system. Chromatographic runs were performed on an H Class Acquity™ UPLC system (Waters, Massachusetts, USA) that was equipped with a tunable UV/VIS detector (Waters).
Chromatographic separation was performed using an Acquity UPLC BEH300 C18 chromatographic column (1.7 μm, 2.1 mm × 150 mm) (Waters) with gradient workflow of 0.4 mL/min of ultrapure purified water (Merck Millipore Co., Darmstadt, Germany) with 0.1% trifluoroacetic acid (TFA) (Sigma-Aldrich, Missouri, USA) and acetonitrile (Mallinckrodt Baker, Pennsylvania, USA) and 0.1% TFA (Sigma-Aldrich). The gradient began with 100% aqueous TFA and decreased gradually to 70% from 2 to 12 min. Then, aqueous TFA was returned to 100% from 12 min to the end of the analysis (20 min). The column temperature and sample cooler were maintained at 30°C and 8°C, respectively. The UV/VIS detector was set to 214 nm for data acquisition, and the analysis was performed using Empower (Waters). Only chromatographic peaks with a capacity factor (k) higher than 1.0 were considered for analysis.

Metabolite analysis was performed on a Waters Acquity ultraperformance liquid chromatography machine with diode array detection (UPLC-DAD) using methods and software described in the Waters Corporation user manual. The manual was adapted for oilseed rape tissue by [34 (link), 35 (link)]. The AccQtag method was used to quantify amino acids and the integration software Empower (Waters Corporation, Milford, USA) was used for analysis. Samples were resuspended in 100 mL distilled water. Subsequently, 5 mL were derivatized using AccQTag Ultra Derivatization Kit, according to the manufacturer’s recommendations. An external standard of 100 mmol/L of each amino acid was run every 10 samples. Quantification of sugars was performed using a gas chromatography-flame ionization detector (GC-FID) System from Agilent Technologies (Santa Clara, CA, USA) according to [36 (link)]. The integrated Agilent software ChemStation Rev.B.04.02 was used for data analysis. Samples were resuspended in 50 mL pyridine (100%) with methoxamine hydrochloride (240 mmol/L), then derivatized with 50 mL MSTFA (N-methyl-N-(trimethylsilyl)trifluoro acetamide) (100%). An external standard containing 400 mmol/L of each sugar, sugar alcohol and organic acid was run every 10 samples.

To perform the simultaneous determination of the seven standard components, an HPLC system (Waters Alliance e2695, Waters Corp., Milford, MA, USA) equipped with an auto sample injector, column oven, pump, and PDA detector (#2998; Waters Corp.) was used. Empower (version 3; Waters Corp) software was used to acquire and process the data. The separation of the seven components was conducted with an analytical column (Gemini C18, 250 × 4.6 mm, 5 μm) (Phenomenex, Torrance, CA, USA) maintained at 30 °C. The mobile phases consisted of 10% (v/v) acetonitrile in 0.2% SDS with 0.02% phosphoric acid (A) and acetonitrile (B). The gradient conditions were as follows: 10–40% B for 0–20 min, 40–50% B for 20–40 min, 50–100% B for 40–50 min, and 100% B for 50–57 min. The injection volume was 10 µL, and the flow rate was 1.0 mL/min.

Four shoot apices (40–80 mg fresh weight) were collected from each step of the cryopreservation procedure. MDA was measured according to Lepage et al. [16 (link)] with a few modifications. Shoot apices were added to 1 ml of 5 % TCA solution (trichloroacetic acid) and 0.1 ml of 20 mM BHT (butylated hydroxytoluene) and incubated at 95 °C for 30 min. After centrifugation at 1000 g for 10 min, an equal volume of 0.25 % thiobarbituric acid (TBA) was added to the supernatant. The reaction mixture was heated at 95 °C for 30 min for the formation of MDA-TBA complex. Aliquots of samples were transferred into microvials, which were placed in an autosampler and were automatically injected into the HPLC reverse-phase system (Hypersil ODS C₁₈ with 5 μm particle size, 4.6 × 100 mm). The MDA (TBA)₂ adduct was eluted using an isocratic mobile phase consisting of 100 % methanol at a flow rate of 0.2 ml/min at 25 °C. MDA was detected by fluorescence at excitation 532 nm and emission 553 nm. Quantification was done using an external standard of 1,1,3,3-tetraethoxypropane (Sigma, St Louis, MO) prepared using the same method as for the samples. Chromatograms were analyzed by EMPOWER (Waters, Germany).

All API concentrations in the swabs and rinse solutions were determined using reversed-phase HPLC (Acquity UPLC System, Waters Corporation, Milford, MA, USA) and the software Empower^® (Waters Corporation, Milford, MA, USA). The detailed methods for each API are summarised in Table 4. The LOQ and LOD of all APIs were determined by regression analysis.