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The development of doping agents based on genes and recombinant proteins, such as growth hormone and erythropoietin, is changing the way that drug testing agencies examine suspect samples in sport. Different strategies are required to detect these macromolecular agents compared with the standard testing for small molecules and their metabolites. Now, there is a shift towards studying the effects of performance-enhancing compounds, rather than the compounds themselves. In the equine field, leading testing agency HFL Sport Science, part of Quotient Bioresearch Ltd. based in Fordham, UK, has begun an extensive study on plasma proteins. Plasma samples are regularly taken in the horseracing world as part of the drug testing regime, so it is a good place to start. However, according to study scientists Chris Barton, Paul Beck, Richard Kay, Phil Teale and Jane Roberts, the proteome of equine plasma is far from complete, so that was their first point of call. Plasma was collected from 40 horses before and after the administration of a testosterone mixed ester preparation (Durateston). Pooled samples were digested with trypsin following reduction and alkylation and the peptides formed were subjected to a preliminary fractionation stage by strong cation exchange. Each fraction was subjected to LC/MS/MS with electrospray ionisation and the proteins were identified by searching the NCBI database with a horse taxonomy filter. A total of 70 proteins were identified, even without depletion of the most abundant ones. This is a 2.5-fold increase in the number of known horse plasma proteins, justifying this step and providing a far broader starting base. One protein highlighted within the set was carboxyesterase, which is present in plasma and is active in hydrolysing exogenous esters such as the mixed steroid ester employed. To devise a targeted analysis method, the peptides identified in the LC/MS/MS study were searched against the annotated horse genome to identify those that were unique to one gene product. Up to two peptides were identified for each protein to form the basis of a multiple reaction monitoring (MRM) assay and four MRM transitions were used to validate the peptide against the protein by one-dimensional LC/MS/MS. Subsequently, only the most intense transition was selected for the final assay in order to decrease the duty cycle and increase the number of MS/MS scans over a peak. The team wanted to employ internal standards for the analysis to increase the reproducibility, but it would be grossly expensive to add one isotopically labelled peptide for each of the 70 proteins. Instead, they chose six that represented proteins relevant to doping studies and synthesised them with carbon-13/nitrogen-15 labelled arginine and lysine. The proteins were added to plasma before protein digestion to give an approximate ratio of 1:1 between labelled and endogenous protein. After some experimentation, the best intra- and inter-batch variations were achieved by normalisation to internal standard peptides. In test studies of plasma spiked with haemoglobin A, plots of the ratio of peak area of analyte:internal standard versus the concentration of added haemoglobin were linear. Thus, protein digestion must reach a stable end point, which is essential for reproducibility. The method was applied to 49 of the 70 proteins that were considered relevant for horse doping. In a 55-minute LC/MS/MS run on plasma from 40 horses collected during routine testing, 76 peptides in total were targeted by MRM. The internal standards displayed a low variance of 13.3% across all samples. After grouping the proteins into type, most of them showed a larger variation at 18.5-26.9%, but these values were regarded as acceptable within a doping control assay. Two protein groups, the haem binding proteins and immune response proteins, showed larger variations of 42.1 and 63.7%, respectively, values considered to be too high for potential biomarker development studies. In the next stage, the researchers studied the variation of protein levels for more than 800 hours after a single intra-muscular injection of the testosterone ester, to identify potential biomarkers of testosterone use. After this time, testosterone levels were still raised. The levels of many proteins remained the same over the whole period. These included the aforementioned carboxyesterase which is apparently not regulated by steroid esters. In fact, many proteins reported to be putative biomarkers of steroid abuse remained unchanged. However, the levels of two proteins, clusterin and leucine-rich alpha-2-glycoprotein, were increased over the course of drug administration. Clusterin, also known as testosterone-repressed prostate message, has been reported to be steroid-regulated in prostate cancer and decreased following testosterone administration. Leucine-rich alpha-2-glycoprotein has been reported to be a biomarker of recombinant human growth hormone in humans, so may be a general marker for anabolic agents across different species. Both of these proteins were still raised even when testosterone levels had diminished near the end of the time course, so they might be able to extend the period of doping detection. The team is now applying the assay to examine the normal protein levels in the broader racing population to cover the effects of gender, age, diet and physical activity, amongst other factors. They are also continuing to monitor the responses to doping agents in the ongoing search for biomarkers of drugs abuse. Related links:
The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.
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