Application of iTRAQ Reagents to Relatively Quantify the Reversible Redox State of Cysteine Residues

Figure 2

(a) ADH-1 homodimer is represented with substrate ethanol and coenzyme A at the active site. Coordinates were downloaded from the Protein Data Bank as a PDB file 2HCY and manipulated with the DeepView free software [18]. Cys⁴⁴ and Cys^277,278 are highlighted, and analysis of ADH-1 amino acid sequence indicates Cys-containing tryptic peptides in red that are amenable to analysis by MS/MS. Cys⁴⁴ forms part of the catalytic centre, and Cys^277,278 is involved in a disulfide. (b) Activity (♦−♦) and free thiols (□- -□) present in ADH-1 after exposure to increasing concentrations of H₂O₂. (c) Ponceau S stain and carbonylation immunoblot of ADH after H₂O₂ exposure; there is equivalent protein loading, but an increase in irreversible carbonylation after exposure to 5 mM H₂O₂ is evident.