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

Abstract

Somatotropins, which are used in cattle for growth and lactating performances, are difficult to reliably detect because no direct method exists. Reversed-phase high-performance liquid chromatography (RP-HLC) coupled to electrospray ionization quadrupole mass spectrometry (ESI/MS) has been developed to separate and characterize the N-terminal peptides resulting from tryptic cleavage of natural and recombinant growth hormones from different species (bovine, porcine, and human) and suppliers. Conditions for tryptic digestion were optimized. This technique was found to be optimal to cleave efficiently the N-terminal peptide of the proteins without releasing too much noise from the matrix. Characterization of the peptides through ESI(+)-MS allowed natural and recombinant growth hormones from bovine and porcine species with N-terminal amino acid sequences differing from one amino acid residue to be discriminated. However, the studied human growth hormones had similar primary sequences that did not permit any discrimination between recombinant and natural forms, thus confirming the known identity of these hormones. Protein digestions with pepsin and chymotrypsin were also compared but were not conclusive due to the too small N-terminal peptides released after proteolysis.

Development of a flow cytometric immunoassay for recombinant bovine somatotropin‐induced antibodies in serum of dairy cows

“…Pinel et al . pointed out that because of the high risk of false positive results in their immunoassay, monitoring anti‐rbST antibodies should be coupled to a confirmatory technique . Surprisingly, Rochereau‐Roulet et al .…”

“…Antibodies are important biomarkers due to their long half‐life and specificity . Although the formation of antibodies to a virtually endogenous protein – only one out of 191 amino acids on the N‐terminal end is replaced – seems surprising at first, it was observed in approximately 70 out of 90 rbST‐treated cows using an enzyme‐linked immunosorbent assay (ELISA) . During human growth hormone treatment, no antibody formation was observed in adults; however in children, antibody formation was observed in up to 68% of the target group .…”

Elimination kinetic of recombinant somatotropin in bovine

“…The characterization of somatotropin by mass spectrometry had already been presented in the past [27,28]. However, these existing studies were limited to standard solutions.…”

A role for metabolomics in the antidoping toolbox?

“…Due to the stringent European regulation regarding the use of hormones for farming, several methods for the detection of hormones in livestock have been developed in the past 20 years 91–95 . Soon, researchers highlighted the necessity to move the screening strategies from direct to indirect approaches, allowing the use of a single sample to detect multiple prohibited substances (reducing the cost for each analysis 96 ), and to broaden the detection window for the hormonal abuse, as derived biological responses have longer turnovers 97–99 .…”