Identification of methionine oxidation in human recombinant erythropoietin by mass spectrometry: Comparative isoform distribution and biological activity analysis
| Autor: | Dariush Norouzian, Reza Ahangari Cohan, M.Reza Khorramizadeh, Mahdi Aminian, Shahram Teimourian, Mohammad Hossein Hedayati |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Gene isoform Spectrometry Mass Electrospray Ionization Oxidative degradation Mass spectrometry Biochemistry 03 medical and health sciences chemistry.chemical_compound Mice 0302 clinical medicine Methionine Tandem Mass Spectrometry hemic and lymphatic diseases Distribution (pharmacology) Animals Humans Protein Isoforms Amino Acid Sequence Recombinant erythropoietin Erythropoietin Chemistry Manufacturing process Electrophoresis Capillary Biological activity General Medicine Recombinant Proteins 030104 developmental biology Oxidation-Reduction 030217 neurology & neurosurgery Biotechnology Chromatography Liquid |
| Zdroj: | Preparative biochemistrybiotechnology. 47(10) |
| ISSN: | 1532-2297 |
| Popis: | Oxidative degradation of human recombinant erythropoietin (hrEPO) may occur in manufacturing process or therapeutic applications. This unfavorable alteration may render EPO inefficient or inactive. We investigated the effect of methionine/54 oxidative changes on the amino acid sequences, glycoform distribution and biological activity of hrEPO.Mass spectrometry was applied to verify the sequence and determine the methionine oxidation level of hrEPO. Isoform distribution was studied by capillary zone electrophoresis method. In vivo normocythemic mice assay was used to assess the biological activity of three different batches (A, B, and C) of the proteins.Nano-LC/ESI/MS/MS data analyses confirmed the amino acid sequences of all samples. The calculated area percent of three isoforms (2-4 of the 8 obtained isoforms) were decreased in samples of C, B, and A with 27.3, 16.7, and 6.8% of oxidation, respectively. Specific activities were estimated as 53671.54, 95826.47, and 112994.93 mg/mL for the samples of A, B, and C, respectively.The observed decrease in hrEPO biological activity, caused by increasing methionine oxidation levels, was rather independent of its amino acid structure and mainly associated with the higher contents of acidic isoforms. |
| Databáze: | OpenAIRE |
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