Figure 1.

Schematic illustration of the approach used to analyze the redox proteome. (1) Samples are lysed in a buffer containing the alkylating reagent d(0) NEM. (2) Excess NEM is removed by desalting, and reversibly oxidized Cys residues are reduced using TCEP. (3) Newly reduced Cys residues are alkylated with the heavy isotopic form of NEM (d5-NEM). (4) Samples are digested using trypsin. (5) Peptides analyzed by LC–MS/MS using QExactive. (6) Label-free quantification of proteins is performed using PEAKS software. (7) Reversible oxidation state of individual Cys redox peptides are relatively quantified using the targeted quantification program Skyline.