Electrophoretic detection of black market myostatin propeptide

Abstract

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.

Annual banned‐substance review 16th edition—Analytical approaches in human sports drug testing 2022/2023

“…Internet-based suppliers have offered myostatin propeptide preparations for research purposes, and means to monitor a potential misuse in sports has been required. Here, Reichel et al presented a comprehensive study on the composition of 12 products (only 9 of which proved to contain the labeled product)and electrophoretic approaches for their detection in human serum and urine 90. Immunoprecipitation followed by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and Western blotting of either 500 μL of serum or 15 mL of preconcentrated urine (ultrafiltration) allowed for LODs of 2.5 and 0.4 ng/mL, respectively, allowing for detecting potential administrations of this protein-based compound in routine doping controls.…”

The 40th Manfred Donike workshop on doping analysis

“…propeptide, 2 selective estrogen receptor modulators, and β 2 -aonists. 3 Also, challenges arising from permitted versus prohibited routes and/or time points of drug administration and consumption of dietary products were addressed.…”

“…Central topics of the 2022 conference can be largely subsumed under analytical aspects concerning steroidal substances and erythropoiesis‐stimulating agents as well as blood transfusions, as well as investigations into the detection, characterization, and synthesis of metabolic products of drugs, including, for example, myostatin propeptide, 2 selective estrogen receptor modulators, and β 2 ‐aonists 3 . Also, challenges arising from permitted versus prohibited routes and/or time points of drug administration and consumption of dietary products were addressed.…”