Electrophoretic detection of black market myostatin propeptide

• Published in: Drug testing and analysis 2022-11, Vol.14 (11-12), p.1812-1824
• Main Authors: Reichel, Christian, Gmeiner, Günter, Thevis, Mario
• Format: Article
• Language: English
• Subjects: Antibodies; black market; Black markets; Blotting, Western; doping control; Drug testing; Electrophoresis, Polyacrylamide Gel; GDF‐8; Immunoprecipitation; myostatin inhibitor; myostatin propeptide; Pharmaceuticals
• ISSN: 1942-7603; 1942-7611
• DOI: 10.1002/dta.3398

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 is presented as well as electrophoretic detection methods for serum and urine. 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 anti‐myostatin 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. Of the 12 black market products investigated, only nine contained the target protein among dozens of other proteins. In the remaining ones, small growth hormone‐related peptides were found by mass spectrometry (e.g., tesamorelin). A shotgun proteomics approach revealed that all nine products contained full length GST‐tags. Two detection methods were developed for serum (500 μL) and urine (15 mL) and were based on immunoprecipitation, SDS‐PAGE, and Western blotting. The obtained LODs were approximately 2.5 ng/mL (serum) and approximately 0.4 ng/mL (urine).