title: Synthesis, characterisation, and mass spectrometric detection of a pegylated EPO-mimetic peptide for sports drug testing purposes.
abstract: Erythropoietin (EPO) and other erythropoiesis-stimulating agents possess a high misuse potential in elite sport due to their ability to increase the oxygen transport capacity, which plays a vital role in enhancing endurance performance. Currently, a new generation of EPO-mimetic peptides is under development from which peginesatide (also referred to as Hematide™), a pegylated homodimeric peptide of approximately 45 kDa with no structural relationship to erythropoietin, is the most advanced drug candidate undergoing phase-III clinical trials. Since preventive doping research aims at the development of detection methods before a drug receives clinical approval, a selective and sensitive assay has to be established knowing that conventional doping control assays for EPO will not succeed in detecting peginesatide. Thus, a pegylated EPO-mimetic peptide simulating the structure and properties of the lead drug candidate peginesatide was synthesised as a model compound for this new class of emerging drugs and characterised by means of gel electrophoresis, matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry, as well as liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) after proteolytic digestion. Based on these results, a mass spectrometric detection method of the product in plasma was developed targeting a pentapeptide fragment after protein precipitation and subtilisin digestion. Its fitness for purpose was evaluated by the determination of selected method characteristics focusing particularly on specificity, recovery (ca. 60%), and limit of detection (1 ng/mL).
authors.affiliation: Institute of Biochemistry - Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany.
authors.forename: Ines
authors.initials: I
authors.lastname: Möller
authors.forename: Andreas
authors.initials: A
authors.lastname: Thomas
authors.forename: Hans
authors.initials: H
authors.lastname: Geyer
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schänzer
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
mesh_terms: D040761:Biomimetic Materials / Q000737:chemistry; D002853:Chromatography, Liquid; D004300:Doping in Sports; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D011092:Polyethylene Glycols / Q000032:analysis* / Q000737:chemistry; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D010455:Peptides; C514771:hematide; D004921:Erythropoietin; D011092:Polyethylene Glycols; C556270:peginesatide