title: Epoetin alpha, epoetin beta and darbepoetin alfa: two-dimensional gel electrophoresis isoforms characterization and mass spectrometry analysis.
abstract: Since 1989 recombinant human erythropoietin (rhEPO) has been used as a drug for the correction of anemia, but the misuse of rhEPO as an ergogenic agent among athletes is a widespread doping practice. As a consequence there is a need for developing reference methods for the detection of rhEPO in biological fluids, and to be able to differentiate the recombinant from the natural protein. Recombinant human erythropoietin differs from its natural counterpart in the glycidic part of the molecule. Three different commercial recombinant products Epoetin alpha (Eprex, Janssen Cilag), Epoetin beta (Neorecormon, Roche) and Darbepoetin alfa (Nespo, Dompè) have been used to evaluate the performance of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) for the separation of isoforms and the identification of the proteins respectively. All the compounds studied were well separated by means of 2-DE: Epoetin alpha and beta focused in the same isoelectric point region giving rise to six and eight spots respectively, whereas Darbepoetin alfa was found in a more acidic zone with two spots. Results obtained with micro high-performance liquid chromatography-electrospray ionization-time of flight (TOF) MS and matrix-assisted laser desorption/ionization-time of flight MS for the three rhEPOs are reported. These preliminary results suggest that by means of 2-DE and MS it should be possible to reveal the presence of rhEPOs for antidoping purposes.
authors.affiliation: Laboratorio Centrale Analisi Biochimico-Cliniche, Azienda Ospedaliera Careggi, Florence, Italy. caldinia@ao-careggi.toscana.it
authors.forename: Anna
authors.initials: A
authors.lastname: Caldini
authors.forename: Gloriano
authors.initials: G
authors.lastname: Moneti
authors.forename: Alessandra
authors.initials: A
authors.lastname: Fanelli
authors.forename: Alessia
authors.initials: A
authors.lastname: Bruschettini
authors.forename: Stefano
authors.initials: S
authors.lastname: Mercurio
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Pieraccini
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Cini
authors.forename: Agostino
authors.initials: A
authors.lastname: Ognibene
authors.forename: Francesca
authors.initials: F
authors.lastname: Luceri
authors.forename: Gianni
authors.initials: G
authors.lastname: Messeri
mesh_terms: D002851:Chromatography, High Pressure Liquid; D000068256:Darbepoetin alfa; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D000068817:Epoetin Alfa; D004921:Erythropoietin / Q000031:analogs & derivatives* / Q000032:analysis* / Q000302:isolation & purification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D013058:Mass Spectrometry / Q000379:methods*; D020033:Protein Isoforms / Q000032:analysis* / Q000302:isolation & purification; D011994:Recombinant Proteins; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D020033:Protein Isoforms; D011994:Recombinant Proteins; C103998:epoetin beta; D004921:Erythropoietin; D000068256:Darbepoetin alfa; D000068817:Epoetin Alfa