NPM-labeling fluorescence assay

The NPM-labeling fluorescence assay was carried out following the method described previously.¹⁰ Standard curves spanning 0 nM to 20 µM were prepared for N-acetyl-L-cysteine (Sigma-Aldrich, Cat# A7250), β-Lactoglobulin A from bovine milk (Sigma, Cat# L7880) and BSA (Sigma-Aldrich, Cat# A3059) in degassed 6 M Guanidine-HCl, phosphate-buffered saline (PBS) pH 7.4. The concentration of the protein samples was adjusted to 8 μM (~170 μg mAb in 150 μL) with degassed 6 M Guanidine-HCl, PBS pH 7.4 so that the expected free sulfhydryl concentration would be between 0.6 and 20 μM. A 0.5 mM NPM (Sigma-Aldrich, Cat# P7908) solution was prepared in DMSO. Ten microliters of 0.5 mM NPM was incubated with 50 µL standard or sample in a black half-area 96-well plate (Corning) at room temperature for 2 h. The incubation was halted by the addition of 10 µL 100 mM HCl. For one replicate of each standard curve, 8 µL of 10 mg/mL pepsin in ddH₂O (Sigma-Aldrich, Cat# P7012) was added to each well and incubated at room temperature for 30 min. Fluorescence was read with an Infinite M200 Pro variable wavelength fluorescence detector (TECAN, Männerdorf, Switzerland) using an excitation wavelength of 330 nm and an emission wavelength of 376 nm.