Probing Cysteine Reactivity in Proteins by Mass Spectrometric EC-Tagging Loı 1c Dayon, Christophe Roussel, and Hubert H. Girault* Laboratoire d’Electrochimie Physique et Analytique, Ecole Polytechnique Fe ´de ´rale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland Received October 28, 2005 The on-line electrochemical tagging (EC-tagging) of cysteine residues in proteins during mass spectrometry is studied to probe the cysteine environment. Benzoquinone probes electrogenerated at a microspray electrode react with the thiol functions of the proteins within a microchannel and the products are analyzed by mass spectrometry. The fundamentals of the technique are discussed, with a focus on the kinetic aspects. The EC-tagging efficiency of the cysteine residues in proteins is used to probe their environment. Experiments with unmodified proteins and their chemically reduced forms highlight the strong effect of the cysteine site reactivity on the tagging efficiencies. This study highlights relevant parameters for such on-line electrochemical derivatization/MS detection strategies. Keywords: cysteine markers • electrochemistry • electrospray • mass spectrometry • protein structures probing Introduction In proteins, cysteine residues are important for metal coordination, catalysis, and protein structure by forming di- sulfide bonds. Moreover, crucial cysteine residues are involved in modulation of protein activity and signaling events via redox reactions, chelation of transition metals and S-nitrosation. Cysteine is also the binding site in human albumin for biological and clinical small molecules such as platinum(II) anticancer drugs. 1,2 The reactivity of cysteine in proteins is complex for reasons such as steric hindrance, charge distribution and solvation. It varies from one protein to another and specific competition with glutathione makes the system even more complex in biological fluids. The antioxidant character of cysteinyl proteins depends highly on the protein structure that for instance prevent the formation of disulfide bonds in albumin or reduce the antioxidant capacity of hemoglobin in comparison to glutathione. 3,4 As nucleophiles, thiols have a reaction rate that depends on the protonation state of the sulfhydryl group. The primary structure of the biomolecule influences the thiol reactivity since the pKa of the thiol is strongly dependent on the charged residues in the vicinity of the cysteine. It has been shown that Michael-type addition of sulfhydryl-containing peptides onto unsaturated groups have higher or lower rates when positive charges and negative charges are respectively in the vicinity, resulting to a decrease or an increase of pK a. 5 In electrospray ionization (ESI) mass spectrometry (MS), the application of an electric field to generate the spray has led to the consideration of emitters as on-line electrochemical flow-cells. Then, the inherent electrochemical aspect of electrospray 6-8 has opened the way to the study of electro- chemically induced reactions 9-12 like rearrangements of bio- logical molecules. 13-15 Moreover, electro-active probes have been developed to chemically derivatize weakly ionisable compounds, 16-21 and to label specific amino acids. 22 Recently, we have developed a polymer micro-ESI emitter comprised of a microband electrode. 23 This micro-flow-cell was shown to be an efficient controlled-current electrochemical flow-cell compared to many commercially available ESI sources. 24 Additionally, the upstream position of the microband electrode within the micromachined flow channel is a major advantage in the electrogeneration of tags to bind to molecules flow- ing above the electrode toward the Taylor cone. The oxidation of hydroquinone derivatives on the microband anode was studied to tag cysteine residues in peptides via a selective 1,4- Michael addition. When controlling both the electrode mass transport and the kinetics of the addition reaction, the ap- plication of on-line counting of cysteines in peptides to the identification of proteins by peptide mass mapping was achieved. 25,26 We present here a method to probe cysteinyl sites in proteins. An analytical kinetic model is developed to predict tagging extents at the end of the microchannel prior to the Taylor cone. We show that the MS measurements of the extent of the EC-tagging reaction depend mainly on the reactivity of cysteine residues in the proteins and not on the ionization properties of adducts. The EC-tagging of -lactoglobulin A (one free cysteine residue) was studied and compared with the multi-tagging of its reduced form (i.e., five free cysteine residues after reduction of the two disulfide bonds). Creatine phospho- kinase and reduced insulin were probed by EC-tagging to complete the study. * To whom correspondence should be addressed. Fax (+41) 21-693-36- 67. E-mail: hubert.girault@epfl.ch. 10.1021/pr050365o CCC: $33.50 © 2006 American Chemical Society Journal of Proteome Research 2006, 5, 793-800 793 Published on Web 02/21/2006