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

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

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Institute of Pharmacy and Translational Medicine, Sechenov First Moscow State Medical University , 2-4 Bolshaya Pirogovskaya Street, 119991 Moscow, Russia.
Anti-Doping Center , Elizavetinskiy per., 10/1, 105005 Moscow, Russian Federation.
NRC Institute of Immunology FMBA of Russia , 24 Kashirskoye Highway, Moscow 115478, Russia.


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.


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

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