Matrix-assisted laser-desorption time-of flight ionisation and high-performance liquid chromatography-electrospray ionisation mass spectral analyses of two glycosylated recombinant epoetins.

The research focuses on the use of mass spectrometric analysis for two recombinant proteins, Eprex and Aranesp, with an aim to produce reference mass spectra and evaluate its usage for controlling doping in equines and canines. The study reveals that deglycosylation reaction can eliminate microheterogeneity from these proteins and they could be detected using MALDI-TOF-MS and HPLC-ESI-MS. Furthermore, the researchers also analyzed eight synthetic reference standards that match peptides produced by the enzymatic cleavage of the two proteins.

Objective of the Research

• The central aim of this research was to conduct mass spectrometric analyses on the recombinant proteins found in Eprex and Aranesp. The researchers’ motivation was to produce reference mass spectra and explore how to improve the method’s application to control equine and canine doping.

Methodology and Findings

• The researchers initiated a deglycosylation reaction, which resulted in a simple and low-chemical-noise reaction. This reaction removed microheterogeneity caused by the post-translational attachment of carbohydrates. This transformation served as proof that these proteins could be detected with the help of MALDI-TOF-MS and HPLC-ESI-MS after deglycosylation. The results also noted that human erythropoietin (hEPO) could be detected using HPLC-ESI-MS once deglycosylated.
• This study’s findings are exceptional as they represent the first published spectra of deglycosylated Eprex and Aranesp, representing a significant step forward in this field of research.
• Researchers performed further analysis on eight synthetic reference standards. These standards matched peptides that are produced owing to the endoproteinase Glu-C enzymatic cleavage of Aranesp and/or Eprex. The analysis of these standards was carried out by ESI-MS and ESI-MS-MS.
• The E12 Glu-C peptide, which is common in both proteins, was detected at a significantly low femtomole-level using gradient nano-HPLC-ESI-MS-MS in a positive ion mode.