Multiplexed LC-MS/MS analysis of horse plasma proteins to study doping in sport.

The research article centres around the creation of a method for discovering potential biomarkers, associated with doping, in horse plasma by analytically monitoring specific peptides from horse proteins.

Explanation of Research

The research was done based on the need to control deceptive practices such as gene and protein doping in sports involving horses. The researchers proposed a potential solution to these doping threats: the development of protein biomarkers which would allow an indirect detection of doping.

• The research revolves around a method that was developed for the discovery of these potential biomarkers. This method monitors proteotypic peptides (peptides that are representative of a particular protein) derived from horse proteins.
• These peptides were initially identified by listing the abundant proteins found in horse plasma. Horse plasma refers to the clear liquid component of horse’s blood that remains after all the blood cells have been removed.
• A method called Liquid Chromatography coupled with Tandem Mass Spectrometry (LC-MS/MS) was then used for the targeted analysis of these identified peptides. This technique involves multiple rounds of mass spectrometry to further isolate specific ions for detection, providing a high degree of specificity.
• Notably, an aspect of this LC-MS/MS which is called multiple reaction monitoring, was employed to observe and study the quantity of 49 proteins in horse plasma in a single run.

Method Validation and Application

Once the proposed method was developed, it was validated and optimized. The method was applied in two main ways:

• Firstly, it was applied to a population of race-horses to study the variance of protein within the population. This would provide a foundational understanding of the normal range of these proteins in healthy, non-doped horses.
• The second application involved studying the longitudinal time courses of horse plasma after the administration of an anabolic steroid. This can be interpreted as studying the changes in the protein levels or their presence over time after the horses had been given a performance-enhancing substance. The purpose of this was to identify any significant changes that could serve as potential biomarkers for doping.

The overall objective of this research was to formulate a method for the discovery of potential biomarkers associated with doping. This would be instrumental in furthering the fight against doping in horse sport by providing a means of detecting unacceptable practices. This early detection could help maintain the integrity of the sport and ensure the welfare of the animals involved.