Discrimination of mammalian growth hormones by peptide-mass mapping

Abstract

Recognition by the legal authorities that growth hormones (GHs) may be abused to improve sporting performance and/or physique has led to the implementation of controls that make it an offence to produce, supply, possess or import and export GHs, with intent to supply, without the authority to do so. A method is described for the discriminatory analysis of human, equine, porcine and bovine GHs for forensic purposes. Peptide-mass mapping by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry following tryptic digestion gave sequence coverages of 97.4%, 93.7%, 94.2% and 90.6% for human, equine, porcine and bovine GHs respectively. The tryptic-mass maps generated were sufficient to discriminate between the four hormones analysed and thus provide unambiguous identification of each individual GH. Identification of the N-terminal peptides of recombinant equine and porcine GHs, which possess additional methionine residues, within the tryptic-mass maps may provide the basis of a test to indicate exogeneous administration rather than endogenous secretion of GH in performance dogs and horses.

Discrimination of Recombinant and Pituitary-Derived Bovine and Porcine Growth Hormones by Peptide Mass Mapping

“…From these examples, evidence is given that unambiguous discrimination of natural and recombinant standard somatotropins differing in one amino acid residue at their N-terminal ends can be achieved through trypsin hydrolysis. A previous tryptic mapping technique has already been successfully employed to identify the species origin of mammalian growth hormones (20,23,24,26); however, these studies were not led with the purpose of discriminating natural from recombinant species. For the first time, we report here a study aimed at discriminating, for a particular species, recombinant and natural hormones.…”

“…Alternative methods have been developed, such as quantification through immunoassay techniques of secondary markers of ST administration such as insulin-like growth factor I (IGF-I): indeed, the main site of ST action is the liver, where they stimulate the production of IGFs (15,17,18). Mass spectrometric peptide mapping has become an established and powerful structural tool for the analysis of proteins (19)(20)(21)(22)(23)(24)(25)(26). This method is based on mass spectrometry analysis of the peptides released after specific enzymatic digestion of a protein.…”

“…Mass spectrometric peptide mapping has become an established and powerful structural tool for the analysis of proteins ( − ). This method is based on mass spectrometry analysis of the peptides released after specific enzymatic digestion of a protein.…”

Identification of Recombinant Equine Growth Hormone in Horse Plasma by LC−MS/MS: A Confirmatory Analysis in Doping Control

“…Parameters of hydrolysis presented here in terms of pH, temperature, and reaction time (pH 7.9, 37 °C, 17 h) are as previously reported to generate GH peptides. [17][18][19][20][21]43 In addition, similar trypsin concentration had already been reported for recombinant human erythropoietin identification in horse plasma by LC-MS/MS. 44 LC-MS/MS Analysis.…”

“…On the basis of differences in amino acid sequence, the molecular weight discrimination by mass spectrometry between endogenous GH and recombinant forms is well documented in several species such as bovine, − pig, and equine. , Moreover, GH detection can be improved in terms of sensitivity and specificity by peptide mass mapping, ,,− involving liquid chromatography−tandem mass spectrometry (LC−MS/MS) for the detection of the respective N-terminal peptides specific to growth hormones. Nonetheless, all data reported were obtained from reference drug standards in clean buffer matrix.…”