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.
Stanley
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SM
The Australian Racing Forensic Laboratory, Randwick Racecourse, Kensington, Sydney, Australia. shawn_stanley@turfclub.com.sg
Poljak
A
A
D002109:Caffeic Acids / Q000032:analysis; D002241:Carbohydrates; D002851:Chromatography, High Pressure Liquid; D000068256:Darbepoetin alfa; D004220:Disulfides / Q000032:analysis; D004300:Doping in Sports; D004921:Erythropoietin / Q000031:analogs & derivatives* / Q000032:analysis*; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents; D036103:Nanotechnology; D011994:Recombinant Proteins; D012015:Reference Standards; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002109:Caffeic Acids; D002241:Carbohydrates; D004220:Disulfides; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D004921:Erythropoietin; D000068256:Darbepoetin alfa; C040048:caffeic acid