On-line cysteine modification for protein analysis: new probes for electrochemical tagging nanospray mass spectrometry

A series of electrogenerated selective electrophiles based on substituted benzoquinones has been characterized as tags for l-cysteine and cysteine residues in proteins. The electrophiles are generated electrochemically from the corresponding hydroquinones. It is shown from mass spectrometry analysis that the electrogenerated benzoquinone can tag the biomolecules. The rate constants pertaining to the addition of l-cysteine onto the electrogenerated benzoquinones have been determined using electrochemical techniques. The substitution patterns have been unraveled leading to the assessment of site-specific rate constants. It is shown that the rate constants are primarily dependent on the electronic nature of the substituents as expressed by the Hammett substitution constant. The apparent tagging yields observed for l-cysteine in nanospray mass spectrometry experiments do not correspond to the yields expected from the electrochemical study, as the ionisation efficiencies are highly dependent on the tag. Finally, the on-line tagging has been tested using β-lactoglobulin A and myoglobin. Based on these results, it is concluded that the tagging reaction is selective towards cysteine when it takes place in the nanospray interface. The results show that the methodology presented can be used for a rapid characterization and identification of reactive sites in biomolecules.