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基因兴奋剂是指以非治疗目的而向运动员体内导入外源基因或细胞等物质,以不正当方法提高运动员成绩的物质或技术。目前随着基因治疗技术的发展,基因兴奋剂被用于竞技体育赛场的潜在问题受到越来越密切的关注,因此对基因兴奋剂检测方法的需求也愈发迫切。本文简要介绍了基因兴奋剂的潜在候选基因、导入途径与载体、基因兴奋剂的危害以及已有基因兴奋剂的检测方法,并对未来基因兴奋剂检测领域急需解决的关键问题进行了讨论。 相似文献
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第29届奥运会即将于2008年8月8日在北京召开,伴随着中国社会的进步和科学、经济的腾飞,百年奥运梦想就要成真,华夏儿女无不为之欢欣。作为分析化学工作者,尤其是色谱工作者,我们在关注奥运本身的体育意义外,还会思考奥运精神以及与此相关的技术问题,其中兴奋剂检测就是一个不得不说的话题。
回顾现代奥林匹克运动的发展史,可以说始终伴随着滥用兴奋剂和反兴奋剂的斗争。随着体育运动的日益商业化,滥用兴奋剂的案例也越来越多。从1967年国际奥委会提出最早的禁用药物名单,到1968年初国际奥委会医学委员会正式宣布了特为法国格勒诺布尔冬奥会兴奋剂检查制定的禁用药物名单。从1999年国际奥委会瑞士洛桑成立世界反兴奋剂机构(WADA),到在联合国教科文组织第33届会议通过《反对在体育运动中使用兴奋剂国际公约》(简称“反兴奋剂国际公约”),再到2007年第三届世界反兴奋剂大会通过修订后的《世界反兴奋剂条例》(将于2009年1月正式生效),兴奋剂的种类越来越多,兴奋剂的检测技术越来越先进,真所谓“道高一尺,魔高一丈”。在某种程度上甚至可以说,分析化学的发展大大促进了反兴奋剂事业的进步,而反兴奋剂的需要,又推动了分析检测技术的更快发展。但无论如何,兴奋剂的滥用不仅严重背离了奥林匹克精神,而且会极大地损害相关运动员和教练员的身心健康。因此,为了配合北京奥运会的召开,为了让读者了解国际上兴奋剂检测研究的现状和我国科学工作者在兴奋剂检测领域的研究成果,也为了北京能办一届“干净”的奥运会,我们特别增设了“兴奋剂检测方法”专栏,共约稿13篇,其中有综述也有研究报告;有传统的检测方法也有最新的毛细管电泳和芯片技术检测兴奋剂的进展;在检测对象方面,既有小分子药物,也有大分子蛋白质;既有传统的药物,也有新的基因类兴奋剂,从不同的侧面反映了当今兴奋剂检测所面临的挑战和新型检测方法与技术大发展的展望。相信读者会从中获得有价值的信息。
非常感谢这么多作者欣然接受我们的邀请,把他们的最新研究结果呈现给我们。特别要感谢北京大学化学学院的白玉博士在稿件审阅和修改过程中所付出的大量劳动。我们愿意将这期专栏作为礼物献给2008年北京奥运会。 相似文献
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Human erythropoietin (hEPO) is a glycoprotein hormone produced primarily by the kidney, which stimulates red blood cell production. Recombinant human erythropoietin (rhEPO), generally produced in Chinese hamster ovary (CHO) cells, can be used as not only a therapeutic protein but also a doping agent in sports. Profiling of EPO glycoforms is a critical means for quality control in pharmaceutical industrial and anti-doping analysis of misuse in sports. However, the existing methods for the analysis of EPO are associated with either time consuming or poor resolution. In this work, a rapid and high-resolution glycoform profiling method was presented based on capillary isoelectric focusing (cIEF) with whole column imaging detection (WCID). Experimental conditions that influence the separation were investigated. Under optimized conditions, rhEPO from three different sources were resolved into distinct populations within 5 min with excellent reproducibility. As compared with existing methods, the presented method exhibited the advantages of speed and high resolution. If combined with an effective sample enrichment step and a much more sensitive WCID version, the method can be a potential alternative for the detection of rhEPO misuse in sports. 相似文献
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Nola H. Yu Emmie N. M. Ho Terence S. M. Wan April S. Y. Wong 《Analytical and bioanalytical chemistry》2010,396(7):2513-2521
Recombinant human erythropoietin (rhEPO), darbepoetin alfa (DPO) and methoxy polyethylene glycol-epoetin beta (PEG-EPO) are
synthetic analogues of the endogenous hormone erythropoietin (EPO). These erythropoiesis-stimulating agents have the ability
to stimulate the production of red blood cells and are commercially available for the treatment of anaemia in humans. These
drugs are understood to have performance-enhancing effects on human athletes due to their stimulation of red blood cell production,
thereby improving delivery of oxygen to the muscle tissues. Although their effect on horses has not been proven, these substances
were thought to be similarly performance enhancing and have indeed been applied covertly to horses. As such, these protein-based
drugs are prohibited by authorities in both human and equine sports. The method officially adopted by the International Olympic
Committee (IOC) and World Anti Doping Agency (WADA) for the confirmation of rhEPO and/or DPO (rhEPO/DPO) in human urine is
based on electrophoresis in combination with Western blotting. A shortcoming of the WADA method is the lack of definitive
mass spectral data for the confirmation of a positive finding. Recently, a liquid chromatography–tandem mass spectrometry
(LC/MS/MS) method for the detection and confirmation of rhEPO/DPO in equine plasma was reported. However, we have not been
successful in achieving the reported sensitivity. This paper presents a method for the detection and confirmation of rhEPO/DPO,
as well as the newly released PEG-EPO, in equine plasma. The procedures involve immunoaffinity extraction using anti-rhEPO
antibody-coated Dynabeads followed by trypsin digestion. The injected extract was further purified and concentrated using
an on-line trap column in the nano-LC system. Detection and confirmation were achieved by monitoring a unique peptide segment
of rhEPO/DPO/PEG-EPO using nano-liquid chromatography–tandem mass spectrometry equipped with a nanospray ionisation source
operated in the selected reaction monitoring mode. rhEPO, DPO and PEG-EPO can be confirmed at 0.1, 0.2 and 1.0 ng/mL, respectively,
in equine plasma. 相似文献
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Estela Giménez Fernando Benavente Carme de Bolós Ernesto Nicolás José Barbosa Victoria Sanz-Nebot 《Journal of chromatography. A》2009,1216(12):2574-2582
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. 相似文献
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Misuse of recombinant human erythropoietin (rhEPO) is a major concern in competitive sports, and the implementation of tests allowing for higher detection rates than what current tests are capable of is required. In this study, a novel lateral flow EPO isoform test kit, EPO WGA MAIIA, is evaluated on the basis of plasma and urine samples obtained from eight healthy males in connection with a 28-day rhEPO injection period. rhEPO was injected every other day during the first 14 days of the study, and the method proved to be 100 % effective in detecting rhEPO in the concomitantly obtained samples. Seven days after the last injection, three positive (>99.99 % confidence limit (CL)) subjects were found. When using 99 % CL as the cut-off limit, six of the eight subjects (75 %) were found to be suspected of doping. Samples obtained 14 and 21 days after the last injection showed no detectable trace of rhEPO. A previous study using indirect methods to determine EPO doping on the same samples indicated only that two of the subjects had suspicious values 7–21 days after the last injection. We propose implementing the easy to-use EPO WGA MAIIA test as an initial screening procedure in anti-doping work to (1) increase the detection rate of potential rhEPO doping athletes and (2) allow for a 10- to 20-fold higher analytical rate than what is possible today. Fig
The lateral flow isoform test, in a dipstick format, can distinguish different types of EPO (blue balls) due to the interaction of their glycosylated structures with the lectin wheat germ agglutinin (WGA), which is the 1st zone to pass. EPO that have succeeded to pass the WGA zone will be captured by EPO-specific antibodies found in the subsequent zone. The percentage of passed EPO is calculated by also measuring the total amount of EPO in the sample using a dipstick with inactive WGA zone. 相似文献
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Bing Gong Irina Burnina Terrance A. Stadheim Huijuan Li 《Journal of mass spectrometry : JMS》2013,48(12):1308-1317
Glycosylation plays a critical role in the in vivo efficacy of both endogenous and recombinant erythropoietin (EPO). Using mass spectrometry, we characterized the N‐/O‐linked glycosylation of recombinant human EPO (rhEPO) produced in glycoengineered Pichia pastoris and compared with the glycosylation of Chinese hamster ovary (CHO) cell‐derived rhEPO. While the three predicted N‐linked glycosylation sites (Asn24, Asn38 and Asn83) showed complete site occupancy, Pichia‐ and CHO‐derived rhEPO showed distinct differences in the glycan structures with the former containing sialylated bi‐antennary glycoforms and the latter containing a mixture of sialylated bi‐, tri‐ and tetra‐antennary structures. Additionally, the N‐linked glycans from Pichia‐produced rhEPO were similar across all three sites. A low level of O‐linked mannosylation was detected on Pichia‐produced rhEPO at position Ser126, which is also the O‐linked glycosylation site for endogenous human EPO and CHO‐derived rhEPO. In summary, the mass spectrometric analyses revealed that rhEPO derived from glycoengineered Pichia has a highly uniform bi‐antennary N‐linked glycan composition and preserves the orthogonal O‐linked glycosylation site present on endogenous human EPO and CHO‐derived rhEPO. Copyright © 2013 John Wiley & Sons, Ltd. 相似文献
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James P. Scarth Cathrin Seibert Pamela R. Brown Phil Teale Gavin J. Beamon Clive M. Pearce Richard A. Sams 《Chromatographia》2011,74(7-8):593-608
Recombinant human erythropoietin (rhEPO) analogues are known to have been used in horse sports for their assumed performance enhancing properties. Recently, several authors have published liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods for confirming the presence of rhEPO analogues in horse plasma. In the current study, an improved LC-MS/MS confirmatory procedure for rhEPO, darbepoetin (DPO) and continuous erythropoietin receptor activator (CERA) in horse plasma was developed and validated. The method was also adapted for and applied to urine samples for the first time. Similar to previously published plasma assays, the methods utilise size exclusion and immunoaffinity extraction prior to tryptic cleavage, enzymatic deglycosylation and LC-MS/MS analysis of the resulting signature tryptic peptides (rhEPO/CERA T5, rhEPO/CERA/DPO T6 and DPO T9). However, the novel application of UPLC chromatography significantly improves the run time of the method compared to nano- or micro-LC and its robustness compared to nano-LC. Furthermore, recombinant canine EPO was found to serve as an effective internal standard, thus allowing confidence in interpretation of the success/failure of every step in the procedure. Limits of detection for confirming the presence of rhEPO, CERA and DPO in plasma were 0.1, 0.25 and 0.05 ng mL?1, respectively, which were equal to or lower than limits achieved using previously published LC-MS/MS based methods. Limits of detection for confirming the presence of rhEPO, CERA and DPO in urine were 0.05, 0.15 and 0.025 ng mL?1 and the analysis of urine samples collected from horses administered rhEPO (Eprex?) or DPO (Aranesp?) demonstrated the use of this matrix as a suitable alternative in situations where plasma is not available. 相似文献
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The test used by anti-doping laboratories to detect the misuse of recombinant erythropoietin (rhEPO) is based on its different migration pattern on isoelectric focusing (IEF) gel compared with the endogenous human erythropoietin (hEPO) that can possibly be explained by structural differences. While there is definitely a need to identify those differences by LC-MS/MS, the extensive characterization that was achieved for the rhEPO was never performed on human endogenous EPO because its standard is not available in sufficient amount. The goal of this study was to develop an analytical method to detect pmol amounts of N-linked and O-linked glycopeptides of the recombinant hormone as a model. Using a nanoflow HPLC-Chip electrospray ionization/ion trap mass spectrometer, the diagnostic ion at m/z 366 of oligosaccharides was monitored in the product ion spectra to identify the four theoretical glycosylation sites, Asn24, Asn38, Asn83 and Ser126, respectively, on glycopeptides 22-37, 38-55, 73-96 and 118-136. With 3 pmol of starting material applied on Chip, only the desialylated N-glycopeptides 22-37 and 38-55/38-43 could be observed, and of all the glycan isoforms, those with the smaller structures were predominantly detected. While the preservation of the sialic acid moieties decreased the detection of all the N-glycopeptides, it allowed a more extensive characterization of the O-linked glycopeptide 118-136. The technique described herein provides a mean to detect glycopeptides from commercially available pharmaceutical preparations of rhEPO with the sensitivity required to analyze pmol amounts of hEPO, which could ultimately lead to the identification of structural differences between the recombinant and the human forms of the hormone. 相似文献
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Lectin affinity chromatography as a tool to differentiate endogenous and recombinant erythropoietins
This work exploits the combination of the lectin affinity chromatography (LAC) with an ultra-sensitive immunochromatographic assay to differentiate several types of erythropoietin (EPO). The chromatographic behaviours of different commercial types of recombinant human EPO (rhEPO), EPO analogues (Aranesp) and urine human EPO (uhEPO) from healthy individuals on eight lectin-Sepharose columns, have been worked out. Results show that when using wheat germ agglutinin (WGA)-Sepharose columns, a careful desorption regime starting with very low concentration (2mM) of the competitive sugar N-acetylglucosamine (GlcNAc) makes it possible to efficiently distinguish endogenous EPO from recombinant EPO and EPO analogues. 相似文献