Mass spectrometric characterization of a biotechnologically produced full-length mechano growth factor (MGF) relevant for doping controls

• A formerly unreported recombinant and non-natural ‘full-length’ MGF was identified and fully characterized
• The MGF derivative is related to IGF-1 isoform 4 with a C-terminal truncation and amino acid substitution
• A misuse of the arguably undetectable substance in elite sport was suggested by anecdotal evidence
• Spiked urine samples were successfully analyzed for MGF at 0.25 ng/mL

ObjectiveSince Goldspink and colleagues identified the expression of the mRNA of an insulin-like growth factor 1 (IGF-1) isoform in response to mechanical stress in 1996, substantial research into the so-called mechano growth factor and its modus operandi followed until today. Promising preclinical results were obtained by using the synthetic, 24-amino acid residues spanning peptide translated from the exons 4–6 of IGF-1Ec (which was later referred to as the mechano growth factor (MGF) peptide), particularly with regard to increased muscle myoblast proliferation. Consequently, the MGF peptide represented a promising drug candidate for the treatment of neuromuscular disorders; however, its misuse potential in sport was also identified shortly thereafter, and the substance (or class of substances) has been considered prohibited according to the regulations of the World Anti-Doping Agency (WADA) since 2005. While various MGF peptide versions have been known to sports drug testing authorities, the occurrence of a ‘full-length MGF’ as offered via illicit channels to athletes or athletes' managers was reported in 2014, arguably being undetectable in doping controls.MethodsAn aliquot of the product was obtained and the content characterized by state-of-the-art analytical approaches including gel electrophoretic and mass spectrometric (top-down and bottom-up) sequencing approaches. Upon full characterization, its implementation into modified routine doping controls using ultrafiltration, immunoaffinity-based isolation, and nanoliquid chromatography-high resolution/high accuracy mass spectrometry was established.ResultsA protein with a monoisotopic molecular mass of 12264.9 Da and a sequence closely related to IGF-1Ec (lacking the signal- and propeptide moiety) was identified. The C-terminus was found to be modified by the elimination of the terminal lysine and a R109H substitution. With the knowledge of the compound's composition, existing doping control assays targeting peptide hormones such as IGF-1 and related substances were assessed as to their capability to detect the full-length MGF. The analyte was detectable at concentrations of 0.25 ng/mL using adapted routine test methods employing immunoaffinity purification followed by nanoscale liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry.ConclusionsA potentially performance enhancing ‘full-length’ MGF derivative was identified and successfully implemented into sports drug testing protocols. Future tests are indicated probing for optimized/dedicated detection methods and assessment of efficacy and elimination kinetics of the substance.