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

J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Mar 5;785(2):205-18. doi: 10.1016/s1570-0232(02)00824-3.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Caffeic Acids / analysis
  • Carbohydrates
  • Chromatography, High Pressure Liquid
  • Darbepoetin alfa
  • Disulfides / analysis
  • Doping in Sports
  • Erythropoietin / analogs & derivatives*
  • Erythropoietin / analysis*
  • Glycosylation
  • Humans
  • Hydrolysis
  • Indicators and Reagents
  • Nanotechnology
  • Recombinant Proteins
  • Reference Standards
  • Spectrometry, Mass, Electrospray Ionization
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Caffeic Acids
  • Carbohydrates
  • Disulfides
  • Indicators and Reagents
  • Recombinant Proteins
  • Erythropoietin
  • Darbepoetin alfa
  • caffeic acid