Double alkylation with maleimide-PEG-biotin: An enrichment method for cysteine redox states.

Cysteine alkylation is widely used in mass-spectrometric based proteomic studies. The oxidation state of each cysteine can be determined by labeling free thiols with one alkylating agent and disulfides with a second alkylating agent that differs in mass from the first. We have developed an improved method utilizing biotin-conjugated maleimides to specifically label cysteine residues in the thiol state and in disulfide linkage. The biotin tag effectuates very efficient enrichment of cysteine containing peptides, greatly increasing sensitivity for those peptides. We also achieve very high recovery of the biotinylated peptides from an avidin column by elution with hexafluoro-2-propanol (HFIP). The method offers improved mapping of the cysteine proteome and its oxidation state.