title: Analysis of recombinant human erythropoietin and novel erythropoiesis stimulating protein digests by immunoaffinity capillary electrophoresis-mass spectrometry.
abstract: In this work, we demonstrate that detection of a specific peptide marker by immunoaffinity capillary electrophoresis-mass spectrometry (IA-CE-MS) could be used to confirm the presence of recombinant human erythropoietin (rhEPO) in solution. Besides the carbohydrate content, the amino acid sequence of novel erythropoiesis stimulating protein (NESP) differs from human erythropoietin (hEPO) at five positions (Ala30Asn, His32Thr, Pro87Val, Trp88Asn, and Pro90Thr). After digesting both glycoproteins in solution by trypsin and PNGase F, two specific proteotypic peptides, EPO (77-97) and NESP (77-97) which differ in three amino acids, were selected as rhEPO and NESP markers, respectively. Both digests and their mixtures were analyzed by IA-CE-MS. The IA stationary phase was prepared from a custom made polyclonal anti-EPO (81-95) antibody immobilized on a solid support of CNBr-Sepharose 4B and was packed in a microcartridge near the inlet of the separation capillary. As the antibody was directed to a synthetic peptide EPO (81-95), only the proteotypic peptide EPO (77-97) was retained. The retained peptide was eluted, separated by electrophoresis and detected by MS. The method was specific to confirm the presence of rhEPO in solution. Although the limits of detection for the peptide marker were similar to those obtained with CE-MS (a few mg/L), these results show the potential of this novel approach to detect in the future rhEPO and its analogues selectively and unambiguously at the levels expected in biological fluids.
authors.affiliation: Departament de Química Analítica, Universitat de Barcelona, Barcelona, Spain.
authors.forename: Estela
authors.initials: E
authors.lastname: Giménez
authors.forename: Fernando
authors.initials: F
authors.lastname: Benavente
authors.forename: Carme
authors.initials: C
authors.lastname: de Bolós
authors.forename: Ernesto
authors.initials: E
authors.lastname: Nicolás
authors.forename: José
authors.initials: J
authors.lastname: Barbosa
authors.forename: Victoria
authors.initials: V
authors.lastname: Sanz-Nebot
mesh_terms: D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D004921:Erythropoietin / Q000032:analysis* / Q000378:metabolism; D006397:Hematinics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014357:Trypsin / Q000378:metabolism
chemical_list: D015415:Biomarkers; D006397:Hematinics; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004921:Erythropoietin; D014357:Trypsin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase