title: Differences in sialic acid O-acetylation between human urinary and recombinant erythropoietins: a possible mass spectrometric marker for doping control.
abstract: Development of a mass spectrometric method for the unambiguous detection of doping with recombinant human erythropoietins (rhEPO) has been attempted for many years. Unfortunately, progress in this field was hampered by the unavailability of highly purified human endogenous EPOs (urinary[uhEPO], serum/plasma EPO)--a prerequisite for generating detailed mass spectrometric glycosylation data necessary for revealing significant differences between uhEPO and rhEPOs. The paper presents the worldwide first analytical data on purified human urinary EPO generated with a high resolution high accuracy mass spectrometer (LTQ-Orbitrap). The focus is on the tryptic O-glycopeptide (E117-R131) and its degree of sialic acid O-acetylation. Data are compared with results obtained from 40 rhEPO pharmaceuticals. It could be demonstrated that the O-glycopeptide of uhEPO (ca 100 IU) contains only trace amounts of mono-acetylated mono-and di-sialylated O-glycans but no other O-acetylated structures and in this respect significantly differs from all rhEPOs. Moreover, Dynepo--a rhEPO previously thought to be not O-acetylated--also contains small amounts of O-acetylations within the O-glycan structure. The results might be useful for anti-doping purposes as well as the development of EPO pharmaceuticals with closer structural similarity to the endogenous hormone.
authors.affiliation: Doping Control Laboratory, AIT Seibersdorf Laboratories, A-2444, Seibersdorf, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Reichel
mesh_terms: D000107:Acetylation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004921:Erythropoietin / Q000737:chemistry* / Q000652:urine*; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D019158:N-Acetylneuraminic Acid / Q000032:analysis*; D011994:Recombinant Proteins / Q000737:chemistry / Q000652:urine; D012709:Serum Albumin / Q000302:isolation & purification; D015813:Substance Abuse Detection / Q000379:methods
chemical_list: D011994:Recombinant Proteins; D012709:Serum Albumin; D004921:Erythropoietin; D019158:N-Acetylneuraminic Acid
keywords: acetylation; biosimilars; human urinary erythropoietin; mass spectrometry; sialic acid