Background Recombinant erythropoietin(rEPO)is commonly used in therapy but may be abused in sports to enhance endurance.In doping analysis,rEPO can be detected in human urine or blood samples at picogram(pg)levels bas...Background Recombinant erythropoietin(rEPO)is commonly used in therapy but may be abused in sports to enhance endurance.In doping analysis,rEPO can be detected in human urine or blood samples at picogram(pg)levels based on its slightly higher molecular weight(MW)than that of endogenous EPO using western blotting(WB).However,a type of variant erythropoietin(VAR-EPO)encoded by the EPO c.577del variant has a similar MW to rEPO,and these 2 molecules cannot be distinguished using conventional analytical methods.A fit-for-purpose method needs to be developed immediately.Methods In this study,we introduced a reverse–normal immunopurification technique for sample pretreatment to remove VAR-EPO from samples to eliminate its interference with rEPO detection.Firstly,a rabbit monoclonal antibody(mAb)that can specifically recognize trace amounts of VAR-EPO with high affinity was generated.Then,using this antibody to enrich VAR-EPO,we developed reverse–normal immunopurification coupled with WB on the purpose of analyzing rEPO in urine and serum samples.Next,the method was fully validated and evaluated using blank samples,spiked samples and rEPO excreted samples.Finally,the identification criteria of rEPO was established.Results A specific anti-VAR mAb with high affinity was developed.Using it,we developed the doping analytical method for rEPO.Our method effectively detects and removes VAR-EPO,enabling accurate rEPO detection.Conclusion A method has already been applied for rEPO confirmation in routine doping analyses.展开更多
Interactions are the essence of all biomolecules because they cannot fulfill their roles without interacting with other molecules. Hence, mapping the interactions of biomolecules can be useful for understanding their ...Interactions are the essence of all biomolecules because they cannot fulfill their roles without interacting with other molecules. Hence, mapping the interactions of biomolecules can be useful for understanding their roles and functions. Furthermore, the development of molecular based systems biology requires an understanding of the biomolecular interactions. In recent years, the mapping of protein-protein interactions in different species has been reported, but few reports have focused on the large-scale mapping of protein-protein interactions in human. Here, we review the developments in protein interaction mapping and we discuss issues and strategies for the mapping of the human protein interactome.展开更多
基金WADA and Beijing Sport University for funding under grant numbers 22B06XZ and 2022YB011
文摘Background Recombinant erythropoietin(rEPO)is commonly used in therapy but may be abused in sports to enhance endurance.In doping analysis,rEPO can be detected in human urine or blood samples at picogram(pg)levels based on its slightly higher molecular weight(MW)than that of endogenous EPO using western blotting(WB).However,a type of variant erythropoietin(VAR-EPO)encoded by the EPO c.577del variant has a similar MW to rEPO,and these 2 molecules cannot be distinguished using conventional analytical methods.A fit-for-purpose method needs to be developed immediately.Methods In this study,we introduced a reverse–normal immunopurification technique for sample pretreatment to remove VAR-EPO from samples to eliminate its interference with rEPO detection.Firstly,a rabbit monoclonal antibody(mAb)that can specifically recognize trace amounts of VAR-EPO with high affinity was generated.Then,using this antibody to enrich VAR-EPO,we developed reverse–normal immunopurification coupled with WB on the purpose of analyzing rEPO in urine and serum samples.Next,the method was fully validated and evaluated using blank samples,spiked samples and rEPO excreted samples.Finally,the identification criteria of rEPO was established.Results A specific anti-VAR mAb with high affinity was developed.Using it,we developed the doping analytical method for rEPO.Our method effectively detects and removes VAR-EPO,enabling accurate rEPO detection.Conclusion A method has already been applied for rEPO confirmation in routine doping analyses.
文摘Interactions are the essence of all biomolecules because they cannot fulfill their roles without interacting with other molecules. Hence, mapping the interactions of biomolecules can be useful for understanding their roles and functions. Furthermore, the development of molecular based systems biology requires an understanding of the biomolecular interactions. In recent years, the mapping of protein-protein interactions in different species has been reported, but few reports have focused on the large-scale mapping of protein-protein interactions in human. Here, we review the developments in protein interaction mapping and we discuss issues and strategies for the mapping of the human protein interactome.
