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Mass spectrometric characterization of a biotechnologically produced full-length mechano growth factor (MGF) relevant for doping controls.
Growth Horm IGF Res ; 24(6): 276-80, 2014 Dec.
Article em En | MEDLINE | ID: mdl-25466910
ABSTRACT

OBJECTIVE:

Since 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.

METHODS:

An 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.

RESULTS:

A 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.

CONCLUSIONS:

A 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.
Assuntos

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Fator de Crescimento Insulin-Like I / Detecção do Abuso de Substâncias / Dopagem Esportivo / Espectrometria de Massas em Tandem Idioma: En Ano de publicação: 2014 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Fator de Crescimento Insulin-Like I / Detecção do Abuso de Substâncias / Dopagem Esportivo / Espectrometria de Massas em Tandem Idioma: En Ano de publicação: 2014 Tipo de documento: Article