title: Analysis of recombinant human erythropoietin glycopeptides by capillary electrophoresis electrospray-time of flight-mass spectrometry.
abstract: Capillary electrophoresis electrospray-mass spectrometry was used to detect and characterize the great variety of O- and N-glycopeptide glycoforms of recombinant human erythropoietin (rhEPO) using an orthogonal accelerating time-of-flight mass spectrometer to obtain their exact molecular masses (CE-TOF-MS). rhEPO was digested with trypsin and Glu-C and analyzed by CE-TOF-MS to detect O(126), N(83), N(24)-N(38) and N(24) and N(38) glycopeptide glycoforms, respectively. Neuraminidase was first used to enhance the detection of the glycopeptides and detect all possible glycoforms contained in each glycosylation site. O(126) and N(83) glycopeptides were extensively characterized. Twelve sialoforms corresponding to 5 different glycoforms were detected in N(83), and for the first time, a sulfated sialoform of this glycopeptide was also detected. In the case of O(126), different sialoforms with different types of sialic acids (Neu5Gc and Neu5Ac) were detected and an estimation of the relative percentage of Neu5Gc versus Neu5Ac was also carried out for this glycopeptide. N(24) and N(38) glycosylation sites were also characterized by CE-TOF-MS after Glu-C digestion and these results permitted to rule out some glycan combinations for N(24)-N(38) glycopeptide glycoforms. This study provided a reliable glycopeptide map of rhEPO and may be regarded as an excellent starting point to analyze rhEPO glycopeptides in biological fluids and detect the use of this hormone in sports.
authors: Department of Analytical Chemistry, University of Barcelona, Barcelona, Spain.
authors: Estela
authors: E
authors: Giménez
authors: Raquel
authors: R
authors: Ramos-Hernan
authors: Fernando
authors: F
authors: Benavente
authors: José
authors: J
authors: Barbosa
authors: Victoria
authors: V
authors: Sanz-Nebot
mesh_terms: D004300:Doping in Sports; D019075:Electrophoresis, Capillary; D004921:Erythropoietin / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D006020:Glycopeptides / Q000032:analysis; D006031:Glycosylation; D006801:Humans; D009439:Neuraminidase / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000378:metabolism
chemical_list: D006020:Glycopeptides; D011994:Recombinant Proteins; D004921:Erythropoietin; D009439:Neuraminidase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin