Based on popularity and previously documented cysteine pK a prediction performance we considered only two methods: PropK a (v3.4) and PypK a (v2.9.4). PropK a is an empirical predictor where the ∆G contributions are described by charge-charge, desolvation, and hydrogen-bonding interactions. Default settings were used when performing the calculation. PypK a uses Monte Carlo simulations to probe the various side chain states and employs DelPhi to resolve the PBE. Default settings were used, except for the salt concentration, which was set according to the experimental setup.