title: Matrix-assisted laser-desorption time-of flight ionisation and high-performance liquid chromatography-electrospray ionisation mass spectral analyses of two glycosylated recombinant epoetins.
abstract: 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.
authors.affiliation: The Australian Racing Forensic Laboratory, Randwick Racecourse, Kensington, Sydney, Australia. shawn_stanley@turfclub.com.sg
authors.forename: S M R
authors.initials: SM
authors.lastname: Stanley
authors.forename: A
authors.initials: A
authors.lastname: Poljak
mesh_terms: 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
chemical_list: D002109:Caffeic Acids; D002241:Carbohydrates; D004220:Disulfides; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D004921:Erythropoietin; D000068256:Darbepoetin alfa; C040048:caffeic acid