Dual reductive/oxidative electrochemistry/liquid chromatography/mass spectrometry: Towards peptide and protein modification, separation and identification

•First proof of principle of a new hyphenated technique: EC/EC/LC/MS.•Combined electrochemical reduction and oxidation in two cells.•Protein disulfide reduction and phenol oxidation are carried out.•Labelling of thiol groups with electrochemically generated quinones.•Separation by RP-LC and subsequent high resolution ESI-MS analysis.

A new hyphenated technique based on on-line dual (oxidative and reductive) electrochemistry coupled to liquid chromatography and high resolution electrospray mass spectrometry is presented. Two liquid streams are combined, with one containing a disulfide, which is reduced to the respective thiol in an electrochemical cell based on a titanium working electrode. The other stream contains phenol, which is electrochemically activated to benzoquinone on a boron-doped diamond working electrode. Upon combination of the two streams, a Michael addition takes places, leading to the covalent coupling of thiol to quinone. In continuous flow, the reaction mixture is transferred into an injection valve and the products are separated by reversed phase liquid chromatography and detected by electrospray-high resolution mass spectrometry. Proof of concept is demonstrated for low molecular mass disulfides and peptides, but further optimization will be required in future work to achieve efficient protein labelling.