Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Darbepoetin alfa (DPO) or Novel Erythropoiesis Erythropoiesis Stimulating Protein (NESP), an analog of recombinant human erythropoietin (rhEPO), is abused as a blood doping agent along with the latter in human sports. This paper describes a new method for unequivocal identification of DPO in human plasma. The analyte was extracted from plasma by immunoaffinity separation with anti-rhEPO antibodies, digested by trypsin followed by PNGase F, and analyzed by liquid chromatography coupled to tandem mass spectrometry. The deglycosylated tryptic peptide, T (9), was employed in DPO identification using liquid chromatographic retention time and major product ions of the T (9) peptide. The limit of detection of this method for DPO was 0.1 ng/mL in plasma, and that of identification was 0.2 ng/mL. This method is definitive and devoid of false positive results, providing "mass fingerprints" for identification of DPO in human plasma samples. Although this method is not applicable to identification of rhEPO in human plasma because it cannot differentiate rhEPO from endogenous EPO, it is the first successful attempt towards establishing a reliable and selective method for definitive identification of exogenously administered EPOs in doping control analyses.
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PMID:Identification of darbepoetin alfa in human plasma by liquid chromatography coupled to mass spectrometry for doping control. 1917 13

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.
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PMID:Analysis of recombinant human erythropoietin and novel erythropoiesis stimulating protein digests by immunoaffinity capillary electrophoresis-mass spectrometry. 1921 28

N-Glycosylation of many glycoprotein drugs is important for biological activity and should therefore be the target of specific and quantitative analytical methods. In this study, we focus on the two N-glycan mapping approaches that are used in pharmacopoeial monograph to analyse N-glycans released from fifteen preparations of recombinant human erythropoietin supplied by ten Chinese manufacturers. Underivatised N-glycans were analysed by high performance anion-exchange chromatography with pulsed amperometric detection and fluorophore-labelled N-glycans were analysed by weak anion-exchange and normal-phase high performance liquid chromatography. N-glycans were also analysed by matrix assisted laser desorption ionisation mass spectrometry. The release of N-glycans by PNGase F was shown to be consistent. Z number, a mathematical expression of the total negatively charged N-glycans composition has provided a convenient way to summarise the complex dataset and it might be suitable for product consistency monitoring. However, this Z number reduces the information of individual acidic N-glycan structure and is also found to be method dependent. Therefore, its use requires clear specification and validation. In this study, we only found weak but positive correlation between the Z number and its bioactivity. Wide range of N-glycans yields were obtained from the fifteen preparations but the significance of their differences is unclear.
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PMID:Glycan analysis of glycoprotein pharmaceuticals: Evaluation of analytical approaches to Z number determination in pharmaceutical erythropoietin products. 2199 7


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