Detection of doping with Myostatin-Propeptide in human urine and blood

Code: 19B05CR

Class S4 of WADA’s Prohibited List 2019 (“Hormone and metabolic modulators”) lists myostatin inhibitors under sub-chapter 4 (“Agents preventing activin receptor IIB activation”). Like follistatin, myostatin-propeptide suppresses signaling of myostatin and subsequently leads to an increase in muscle mass and loss of body fat. In serum, >70% of myostatin is bound to myostatin-propeptide and thus myostatin-propeptide regulates skeletal muscle mass, i.e. if myostatin-propeptide is administered, more myostatin will be inhibited and then more muscle mass will be developed. Myostatin-propeptide is a glycoprotein containing one N-glycosylation site and 243 amino acids. Typical concentrations in serum and plasma are in the range of ng/mL.

So far, no approved myostatin-propeptide pharmaceuticals are available. Nevertheless, myostatin-propeptides can be bought on the black market for “research purposes”. They are labelled either “MyoPro”, “HMP”, “Myostatin-Propeptide (HMP)”, or erroneously “GDF-8” and “Myostatin”. All of these proteins are expressed in E. coli and hence lack the characteristic glycosylation of human endogenous myostatin-propeptide. This fact will be exploited in order to detect doping with myostatin-propeptide. After immunoaffinity purification (serum, urine), myostatin-propeptide will be separated by electrophoresis (SDS- or IEF-PAGE) and detected by Western blotting. Due to the missing glycosylation, “black market” products will not only differ in molecular mass but also isoelectric point (pI) from endogenous myostatin-propeptide.

Main Findings

Myostatin propeptide is prohibited according to chapter S4 of the “WADA 2022 List of Prohibited Substances and Methods.” So far, no approved myostatin-propeptide pharmaceuticals are available. Nevertheless, myostatin-propeptides can be bought on the black market for “research purposes.” A study on black market myostatin propeptide products was performed and electrophoretic detection methods for serum and urine were develeoped. Out of the 12 tested products, only nine actually contained the protein. Separation by SDS-PAGE revealed that the nine products were relatively impure and that the main compound had a much higher mass (approximately 54–55 kDa) than expected (approximately 33 kDa). Further analyses by mass spectrometry showed that the elevated molecular mass was due to the presence of a full length GST-tag on the propeptide. The developed detection method for serum is based on immunoprecipitation (IP) followed by SDS-PAGE and Western blotting. In total, three antimyostatin propeptide antibodies were tested. All of them were well suited for either IP or immunoblotting. The final protocol applies a biotinylated polyclonal antibody, streptavidin-coated magnetic beads, and a monoclonal detection antibody. For a sample volume of 500 μL serum, the detection limit of the method is approximately 2.5 ng/mL. The urine method applies a commercial ELISA for IP and performs with a limit of detection (LOD) of approximately 0.4 ng/mL. Furthermore, practically all currently available myostatin propeptide standards were also investigated. Due to the significant molecular mass difference of the black market products, an unambiguous differentiation from endogenous myostatin propeptide is possible. Publication: Reichel C, Gmeiner G, Thevis M. Electrophoretic detection of black market myostatin propeptide. Drug Test Anal. 2022;14(11-12):1812-1824.