Bromomaleimides react rapidly and selectively with cysteine to afford thiomaleimides which can be cleaved with a phosphine to regenerate the cysteine or treated with a base to afford dehydroalanine.

Site-selective dual labelling is affected using local protein microenvironment to control the fate of reaction between cysteine residues and 2,5-dibromohexanediamide.

A mild and highly efficient, reversible protein biotinylation method using a hydrolytically stable linker and mild disassembly conditions is described.

Herein we describe a rapid, simple method for dual modification of DARPins by introduction of cysteine mutations at specific positions that results in a vast difference in their thiol nucleophilicity, allowing for sequential modification.