Exploration Of Ubiquitination Modification Of Doping Growth Hormone Based On High-resolution Mass Spectrometry

Objective:Growth hormone(GH)and its analogs,as a kind of prohibited substances for athletes,have attracted continuous attention due to their high frequency of abuse and difficulty in screening for antidoping purpose.Currently,the detection methods of growth hormone for doping control include direct detection and indirect detection methods.The direct detection method is a kind of isoform differential immunoassays which can be used to distinguish between the proportions of hGH isoforms found under normal physiological conditions and those found after rhGH injection.This method has high sensitivity,but the detection window is short.Besides,positive results can only be found within 24 hours.Indirect testing determines a positive result by an abnormally high concentration of two biomarkers.This method has a long detection window,but the threshold is high,and there is a certain false negative rate.Both methods use population-based decision limits.Here we start from the essence of doping GH,expressed by exogenous cells to distinguish between human endogenous GH and recombinant human growth hormone(rhGH)drugs.The amino acid sequence of endogenous GH is the same as rhGH drugs.This study starts from post-translational modifications(PTM).The existence of PTM judged from the possible differences between rhGH drugs and endogenous GH.Methods:This thesis consists of two studies.The human experimentation in healthy subjects has been approved by the ethics committee of Shanghai University of Sport.Study 1:Exploration of rhGH ubiquitination modificationDifferent peptides are obtained by reducing alkylation and enzymatic hydrolysis in solution before using high-resolution mass spectrometry technology.The proteins are identified from bottom to top to explore PTM of rhGH drugs.PTM of rhGH drugs are explored and matched using different software and confirmed with standards.The results of endogenous GH PTM are preliminarily obtained through literature search.Study 2:Exploring the ubiquitination of endogenous GH in humans under various physiological statesBy sample size calculation,24 healthy subjects are included.We take blood and obtain serum samples from participants in the sleeping at night and running for 6 km groups.Ubiquitinated GH is detected with serum in different physiological states.Results:In study 1,different brands of rhGH drugs are detected to contain the same ubiquitination modification of 2 sites,which is matched by different software and standards.Through literature search,human serum contains ubiquitinated GH.GH with ubiquitination in the human body alone cannot be judged as positive for doping GH.In Study 2,mass spectrometry results are matched with different software and compared with standards.Human GH in multiple physiological states have the same ubiquitin modification sites as rhGH drugs.Conclusion:(1)Through "bottom-up" protein detection strategy,multi-site ubiquitination modification is found for the first time in two marketed rhGH drugs.Besides,the K96 site ubiquitination modification exists in both rhGH drugs.(2)Endogenous GH in human serum under a variety of physiological states can also detect a low proportion of ubiquitination modifications.(3)GH ubiquitination is commonly found in endogenous GH and rhGH drugs.Subsequent attempts can be made to investigate its possibility of use in stimulant screening by evaluating the law of its concentration change.