Analytica Chimica Acta 515 (2004) 261–269 Derivatisation of peptides with osmium tetroxide, 2,2 ′ -bipyridine: capillary electrophoretic and MALDI–TOF mass spectrometric study O. Šedo a , S. Billová b,c , E.M. Peña-Méndez a,d , E. Paleˇ cek b , J. Havel a,∗ a Department of Analytical Chemistry, Faculty of Science, Masaryk University, Kotlᡠrská 2, 61137 Brno, Czech Republic b Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 61265 Brno, Czech Republic c Masaryk Memorial Cancer Institute, Žlutý kopec 7, 65653 Brno, Czech Republic d Department of Analytical Chemistry, Nutrition and Food Science, University of La Laguna, Campus de Anchieta, 38071 La Laguna, Tenerife, Spain Received 6 August 2003; received in revised form 29 January 2004; accepted 24 March 2004 Available online 18 May 2004 Abstract Site-specific chemical modification is a useful technology in characterisation of proteins, but the number of chemical probes of the protein structure reacting with proteins under mild conditions in aqueous solutions is rather limited. Here we studied the reaction of osmium tetroxide, 2,2 ′ -bipyridine (Os,bipy) with several peptides using capillary zone electrophoresis (CZE) and matrix-assisted laser desorption-ionisation–time-of-flight mass spectrometry (MALDI–TOF MS). Both techniques showed formation of a stable complex of Os,bipy with tryptophan residues. In CZE peaks with different migration times and UV-Vis spectra were observed. MALDI–TOF MS showed the formation of a product with characteristic isotopic pattern corresponding to the presence of osmium atom. Oxidation of cysteine and me- thionine side chains to cysteic acid and methionine sulfone by Os,bipy was detected by CZE and confirmed by MALDI–TOF and post-source decay (PSD) mass spectra. PSD showed specific shifts of molecular weights of the peptides and their fragments after the derivatisation. We believe that Os,bipy may become a useful agent in the characterisation of proteins. © 2004 Elsevier B.V. All rights reserved. Keywords: Peptide derivatisation; Osmium tetroxide; 2,2 ′ -Bipyridine; CZE; MALDI–TOF MS; PSD 1. Introduction After a successful genome project, the proteome is now one of the most important projects of mankind. The mass spectrometric methods, especially matrix-assisted laser desorption-ionisation–time-of-flight mass spectrometry (MALDI–TOF MS), are frequently applied in the protein structure determination [1]. Site-specific chemical modifi- cation is often used in protein analysis [2–6]. A number of chemical agents are available reacting more or less specifically with individual amino acid residues but only few of them retain their reactivity and/or specificity under conditions where most of the proteins keep their native structures. In this paper we use a new chemical agent form- ing a stable osmium-containing adduct with peptides under ∗ Corresponding author. Tel.: +420-5-41-12-95-68; fax: +420-5-41-21-12-14. E-mail address: havel@chemi.muni.cz (J. Havel). conditions close to physiological and show mass spectra of these adducts. About 20 years ago we introduced several osmium tetrox- ide complexes with nitrogen ligands, including osmium tetroxide, 2,2 ′ -bipyridine reagent (Os,bipy) as probes of the DNA structure (reviewed in Refs. [7–9]), which have been successfully applied in vitro and in cells at single nucleotide resolution [8,9]. Os,bipy forms stable adducts with thymine and cytosine in single-stranded, but not in perfectly double-stranded DNA. We also showed that Os,bipy-modified DNA (DNA-Os,bipy) produced character- istic voltammetric signals at mercury electrodes [9] allowing determination of DNA-Os,bipy at the submicrogram level. In contrast to nucleic acids, reactions of osmium tetroxide com- plexes with proteins have been studied very little [10–13] despite the fact that these bulky osmium compounds, react- ing covalently with proteins in mild conditions, are good candidates for probes of the protein structure. Mechanism of the reaction of 1-methyl--N-acetyl-dl-tryptophan with 0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.03.066