(A) Assessment of the decrease in free thiol content over time using Ellman’s assay for γS-wt (black), γS-3 (yellow), γS-5 (blue), γS-7 (green), and γS-9 (pink). Measurements were performed in triplicate. Data are represented as mean ± SD. (B) Size-exclusion chromatography in line with multiangle light scattering (SEC-MALS) conducted on each variant after aging for 1 month. High molecular weight (HMW) peaks are seen in γS-5, γS-7, γS-9. (C) Intact mass spectra of each aged variant after reaction with iodoacetamide (IAM) and subsequent reduction with dithiothreitol (DTT). The average free thiol content per protein molecule weighted by the peak intensity decreases with increasing deamidation.