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

Mass spectrometric analyses of the recombinant proteins in Eprex® and Aranesp® were undertaken with the goal of producing reference mass spectra and evaluating strategies to improve its applicability as a method for equine and canine doping control of these substances. A simple, low chemical noise deglycosylation reaction removed microheterogeneity due to post-translational carbohydrate attachment and both proteins were detectable using MALDI-TOF-MS. Deglycosylated human erythropoietin (hEPO) was also detected using HPLC–ESI-MS. This is the first time that spectra of deglycosylated Eprex and Aranesp have been published. Eight synthetic reference standards, which match peptides produced by endoproteinase Glu-C enzymatic cleavage of Aranesp and/or Eprex, were analysed by ESI-MS and ESI-MS–MS. The E12 Glu-C peptide, common to both proteins, was detected at the low femtomole-level using gradient nano-HPLC–ESI-MS–MS in the positive ion mode.