Varioscan plate reader

Protein was extracted from wholemeal flour without defatting (20 mg, n = 4) of malting barley grain at each time point using 400 µL (20 µL/mg) of 8 M urea and 2% (w/v) dithiothreitol (DTT) buffer. The suspension was vortexed and sonicated (Soniclean Ultrasonic Cleaner 25HD, 650W, 43 kHz) for 5 min at room temperature. The samples were incubated on a shaker block (Thermo-Scientific, AUS) at 400 rpm for 45 min at room temperature (RT). The solutions were centrifuged for 15 min at 20,800 x g, and the protein extracts (supernatant) were used for subsequent reduction, alkylation, and filter-aided (MWCO 10kDa) digestion using trypsin (Promega, NSW, AUS) as per established methods (Colgrave et al., 2016 (link)). Protein concentrations were determined via a Varioscan plate reader (Thermo-Scientific, AUS) using Bradford protein assay (California, USA) following the manufacturer’s protocol with dilutions and BSA standard curve. The tryptic peptides were re-suspended in ddH₂O containing 1% formic acid with the addition of iRT reference peptide solution (1 pmol/μL; Biognosys, Zurich, CHE) for subsequent LC-MS/MS analysis.

Proteins on nanoparticles were quantified by using a Pierce BCA Protein Assay Kit (Thermo Scientific). For SNPs and PsNPs, the proteins were quantified without stripping from nanoparticles. The pellet of washed nanoparticle–corona complexes was resuspended in 50 µl of PBS. About 400 µl of working reagent (copper solution) was added to the samples and standard solutions and then incubated for 30 min at 37 °C. The samples were centrifuged at 20,000 × g for 30 min. About 200 µl of each supernatant was transferred into a 96-well plate, and the absorbance at 562 nm was measured using a Varioscan plate reader (Thermo Scientific). In order to evaluate the degree of nanoparticle interference, two control samples were performed: pristine nanoparticles in PBS and in BSA standard solutions⁴⁹ . For nanoparticles that interfere with the BCA assay, the proteins should be eluted first from nanoparticles by the protocol that was explained before in the “Azide modification of HC proteins” section and then analyzed by BCA assay.

Enzymatic activities of cathepsins B, D, and G were determined fluorometrically in snap-frozen proliferating (n = 3), involuting (n = 3), and involuted (n = 3) IH samples from nine patients of the original cohort, using cathepsin B activity assay kit (Calbiochem), cathepsin D activity assay kit (Abcam), and cathepsin G activity assay kit (Abcam), respectively. All steps of the procedure were performed according to the manufacturers’ protocol. Fluorescence was measured in 96-well plate format using the Varioscan plate reader (ThermoFisher).