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

Abstract

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.

Annual banned‐substance review: Analytical approaches in human sports drug testing

“…Focusing on EPO‐Fc fusion proteins, Mesonszhnik et al presented an LC–MS/MS‐based test method, targeting peptidic structures of non‐natural amino acid sequence composition originating from the fusion regions of EPO and the fragment crystallizable (Fc) portion . Following an extensive bottom‐up sequence characterization conducted with a variety of complementary peptidases, a potential target peptide obtained through Lys‐C hydrolysis was identified.…”

Isolation of Branched Chain Aminoacids (Valine, Leucine and Arginine) Using Maghemite Particles

“…Nowadays, various methods are developed for branched chain amino acids (BCAAs) such as valine, leucine and arginine determination. Most of them are based on chromatography (GC, LC, UHPLC) [8] with tandem of mass spectrometry (MS) [9], IEC [10] or CE [11]. In our paper, we decided to use ion exchange chromatography with post-column ninhydrin derivatization and VIS detector for valine, leucine and arginine detection [10].…”