Characterization and Detection of Erythropoietin Fc Fusion Proteins Using Liquid Chromatography-Mass Spectrometry

J Proteome Res. 2018 Jan 5;17(1):689-697. doi: 10.1021/acs.jproteome.7b00739. Epub 2017 Dec 19.


Erythropoietin Fc (EPO-Fc) fusion proteins are potential drug candidates that have been designed for the treatment of anemia in humans by stimulating erythrocyte production. Such compounds can be considered performance-enhancing agents that may be used by athletes in endurance sports. This study describes the primary structure of commercially available EPO-Fc based on comprehensive liquid chromatography coupled with mass spectrometry (LC-MS) analysis. A bottom-up approach and the intact molecular weight (MW) 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. We demonstrated that "spacer peptides" can be used in the determination of EPO-Fc fusion proteins in biological samples using common LC-tandem MS methods.

Keywords: EPO-Fc; IdeS; bottom-up; fusion protein identification; intact mass spectrometry analysis; middle-up; signature peptide.

MeSH terms

  • Amino Acid Sequence
  • Chromatography, Liquid
  • Erythropoiesis / drug effects
  • Erythropoietin / genetics*
  • Humans
  • Immunoglobulin Fc Fragments / genetics*
  • Peptide Hydrolases / metabolism
  • Recombinant Fusion Proteins / analysis*
  • Recombinant Fusion Proteins / genetics
  • Tandem Mass Spectrometry


  • EPO protein, human
  • Immunoglobulin Fc Fragments
  • Recombinant Fusion Proteins
  • Erythropoietin
  • Peptide Hydrolases