Label-free Proteomics for Discovering Biomarker Candidates for Controlling Krypton Misuse in Castrated Horses (Geldings).

This research paper focuses on the identification and monitoring of biomarker candidates in horses specifically for doping control in equine sports using label-free quantitative proteomics. The team successfully utilized this approach to detect and differentiate horses administered with krypton, a potential doping agent, from those that were not treated, for a period of up to 13 days.

Experimental design

• The researchers used label-free quantitative proteomics – a method that allows quantitation of proteins without the need for an isotopic or isobaric tag – to profile plasma proteins in horses following the administration of krypton (Kr).
• Krypton is a potential doping agent as it can stimulate erythropoiesis, the production of red blood cells, hence potentially improving the performance of race horses.
• For the experiment, they utilized nanoliquid chromatography-high-resolution mass spectrometry, a technique used for the identification and quantitation of proteins.

Mass Spectral Library and Data Collection

• An in-house mass spectral library was created featuring 1121 proteins. This was compiled using samples from geldings (castrated horses) used in the administration trial as well as those in training.
• The data was collected by using a data-independent acquisition method. This method is used to quantify a wide range of plasma proteins across samples from the administration trial.

Results and Analysis

• Statistical analyses led to the identification of 83 potential biomarker candidates. These biomarkers showed a successful differentiation between krypton-administered horses and untreated ones, detecting exposure over a period of 13 days.
• This detection time is significantly more than the 1-hour detection capability of plasma krypton (Kr) through headspace gas chromatography-tandem mass spectrometry.
• The study also applied bioinformatics to enrich proposed biomarker candidates and showed a relationship with complement activation and iron metabolism.
• One of the biomarker candidates linked with iron metabolism, transferrin receptor protein 1, demonstrated an upregulation after krypton administration, further validating the study’s findings.
• The researchers verified these results by selected reaction monitoring of corresponding peptides.

Conclusion

• This research successfully showed that the application of label-free quantitative proteomics can be used as an effective strategy to propose plasma protein biomarkers for doping control in equine sports.
• The research data has been made openly accessible via ProteomeXchange with identifier PXD017262.