The analytical approach for detection of carbamylated erythropoietin for doping control purposes

Erythropoietin (EPO) has protective effects in several tissues and could be used for therapeutic purposes, but the doses of EPO that can be beneficial in case of hypoxic-ischemic conditions due to overinduced erythropoiesis could be detrimental in treated patients. Carbamylation of erythropoietin maintains the tissue-protective effects of EPO but without erythropoietic effects. Carbamylated EPO (CEPO) is listed in WADA Prohibited List in class S2 as "Innate repair receptor agonists." The CEPO was synthesized using the method described previously. Digestion with endoproteinase Lys-C was used to distinguish rhEPO from CEPO. The digested samples containing recombinant EPO, urinary EPO (uEPO), or CEPO were analyzed by the SAR-PAGE method (sarcosyl polyacrylamide gel electrophoresis-PAGE). Endoproteinase Lys-C breaks the peptide chains of lysine. Lysine residues, converted to homocitrulline by carbamylation, cannot be cleaved by endoproteinase Lys-C. Therefore, the CEPO protein chain remained unchanged in contrast to rhEPO and uEPO, which allows for easily differentiation of them.