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.
Copyright 2002 Elsevier Science B.V.
Publication Date: 2003-01-30 PubMed ID: 12554133DOI: 10.1016/s1570-0232(02)00824-3Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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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.
Cite This Article
APA
Stanley SM, Poljak A.
(2003).
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, 785(2), 205-218.
https://doi.org/10.1016/s1570-0232(02)00824-3 Publication
Researcher Affiliations
- The Australian Racing Forensic Laboratory, Randwick Racecourse, Kensington, Sydney, Australia. shawn_stanley@turfclub.com.sg
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
Citations
This article has been cited 2 times.- Gianoncelli A, Bonini SA, Bertuzzi M, Guarienti M, Vezzoli S, Kumar R, Delbarba A, Mastinu A, Sigala S, Spano P, Pani L, Pecorelli S, Memo M. An Integrated Approach for a Structural and Functional Evaluation of Biosimilars: Implications for Erythropoietin. BioDrugs 2015 Aug;29(4):285-300.
- Harvey DJ. Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2003-2004. Mass Spectrom Rev 2009 Mar-Apr;28(2):273-361.
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