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)

The structures of the N-linked oligosaccharides of the urinary erythropoietin (u-EPO) purified from urine of aplastic anemic patients were analyzed and compared with those for recombinant erythropoietin (r-EPO) prepared with baby hamster kidney (BHK) cells. Asparagine-linked neutral oligosaccharides were released from each EPO protein by N-oligosaccharide glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by high-performance liquid chromatography (HPLC) on an ODS silica column. More than 8 and 13 kinds of oligosaccharide fractions for u-EPO and r-EPO (BHK), respectively, were completely separated by the one-step HPLC procedure. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amide-silica column. Furthermore, high-resolution proton nuclear magnetic resonance (1H NMR) spectroscopy and methylation analyses were carried out in the case of r-EPO (BHK).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparative structural study of N-linked oligosaccharides of urinary and recombinant erythropoietins. 317 69

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