Optimized Detection of EPO-Fc in Human Biological Fluids

Recombinant erythropoietin (rhEPO) is often used as “doping agent” by athletes in endurance sports to increase blood oxygen capacity. Some strategies improve the pharmacological properties of erythropoietin (EPO) through the genetic and chemical modification of the native EPO protein. The EPO-Fcs are fusion proteins composed of monomeric or dimeric recombinant EPO and the dimeric Fc region of human IgG molecules. The Fc region includes the hinge region and the CH2 and CH3 domains. Recombinant human EPOs (rhEPO) fused to the IgG Fc domain demonstrate a prolonged half-life and enhanced erythropoietic activity in vivo compared with native or rhEPO.

Drug-testing agencies will need to obtain primary structure information and develop a reliable analytical method for the determination of EPO-Fc abuse in sport. The possibility of EPO-Fc detection using nanohigh-performance liquid chromatography−tandem mass spectrometry (HPLC−MS/MS) was already demonstrated (1). However, the prototyping peptides derived from EPO and IgG are not selective enough because both free proteins are naturally presented in human serum. In a recent publication, researchers describe the effort to identify peptides covering unknown fusion breakpoints (later referred to as “spacer” peptides; 2). The identification of “spacer” peptides will allow the confirmation of the presence of exogenous EPO-Fc in human biological fluids.

A bottom-up approach and the intact molecular weight measurement of deglycosylated protein and its IdeS proteolytic fractions was used to determine the amino acid sequence of EPO-Fc. Using multiple proteases, peptides covering unknown fusion breakpoints (spacer peptides) were identified.

Results indicated that “spacer peptides” could be used in the determination of EPO-Fc fusion proteins in biological samples using common LC−tandem MS methods.


  1. Reichel, C. et al. (2012) Detection of EPO-Fc fusion protein in human blood: screening and confirmation protocols for sports drug testing.
    Drug Test. Anal. 4, 818−29.
  2. Mesonzhnik, N. et al. (2017) Characterization and Detection of Erythropoietin Fc Fusion Proteins Using Liquid Chromatography−Mass Spectrometry.
    J. of Proteome Res. 17, 689-97.