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Drug testing and analysis2021; 13(5); 1034-1047; doi: 10.1002/dta.2988

Label-free proteomics for discovering biomarker candidates of RAD140 administration to castrated horses.

Abstract: Selective androgen receptor (AR) modulators (SARMs) are potent anabolic agents with a high potential of misuse in horseracing and equestrian sports. In this study, we applied label-free proteomics to discover plasma protein biomarkers in geldings (castrated horses) after administration with a popular SARM named RAD140. Tryptic peptides were prepared from plasma samples and analyzed by nano-flow ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (nano-UHPLC-HRMS/MS) using data-independent acquisition (DIA) method. Orthogonal projection on latent structure-discriminant analysis (OPLS-DA) has led to the development of a predictive model that could discriminate RAD140-administered samples from control samples and could also correctly classify 18 out of 19 in-training horses as control samples. The model comprises 75 proteins with variable importance in projection (VIP) score above 1. Gene Ontology (GO) enrichment analysis and literature review have identified upregulation of AR-regulated clusterin, and proteins associated with inflammation (haptoglobin, cluster of differentiation 14 [CD14], and inter-alpha-trypsin inhibitor heavy chain 4 [ITIH4]) and erythropoiesis (glycosylphosphatidylinositol-specific phospholipase D1 [GPLD1]) after RAD140 administration. Their changes were confirmed by selected reaction monitoring (SRM) experiments. Similar effects have been reported by the use of androgens and other SARMs. This is the first reported study that describes the use of a proteomic biomarker approach to detect horses that have been administered with RAD140 by applying label-free proteomic profiling of plasma samples. These results support the concept of a biomarker-driven approach to enhance the doping control of RAD140 and potentially other SARMs in the future.
© 2020 John Wiley & Sons, Ltd.
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Publication Date: 2021-01-05 PubMed ID: 33277807DOI: 10.1002/dta.2988Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article presents a study on the use of label-free proteomics to discover plasma protein biomarkers in castrated horses administered with RAD140, a popular selective androgen receptor (SARM). The researchers used this technique to identify plasma proteins that change in response to RAD140 administration, marking a first in exploring such detection methods for doping control in horses.

Research Methodology

  • The researchers conducted the study on geldings which are castrated horses. These horses were administered with a SARM known as RAD140.
  • To discover plasma protein biomarkers, they prepared tryptic peptides from plasma samples from these horses. These peptides were then analyzed through a method known as nano-flow ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (nano-UHPLC-HRMS/MS) that used a data-independent acquisition (DIA) method.
  • The researchers developed a predictive model using OWL-DL (Orthogonal Projection on Latent Structure-Discriminant). This model has been found to be effective in distinguishing samples of horses administered RAD140 from control samples.

Findings

  • The predictive model was made up of 75 proteins that had a variable importance in projection (VIP) score above 1.
  • Through Gene Ontology (GO) enrichment analysis and a review of the existing literature, it was discovered that administration of RAD140 led to the upregulation of AR-regulated clusterin and proteins associated with inflammation and erythropoiesis.
  • Such proteins included haptoglobin, cluster of differentiation 14 (CD14), and inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) that are associated with inflammation and glycosylphosphatidylinositol-specific phospholipase D1 (GPLD1) that is linked to erythropoiesis.
  • The observed changes were further confirmed through selected reaction monitoring (SRM) experiments.

Significance and Application

  • This study marks the first evidence of the use of a proteomic biomarker approach to detect the administration of RAD140 in horses.
  • Such an approach is important in equestrian sports and horseracing where SARMs like RAD140 have high misuse potential as doping agents.
  • The biomarker-driven approach to enhance doping control of RAD140 and potentially other SARMs is supported by this study.

Cite This Article

APA
Cheung HW, Wong KS, To NS, Bond AJ, Farrington AF, Prabhu A, Curl P, Wan TSM, Ho ENM. (2021). Label-free proteomics for discovering biomarker candidates of RAD140 administration to castrated horses. Drug Test Anal, 13(5), 1034-1047. https://doi.org/10.1002/dta.2988

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 13
Issue: 5
Pages: 1034-1047

Researcher Affiliations

Cheung, Hiu Wing
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Wong, Kin-Sing
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
To, Ning Sum
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Bond, Amanda J
  • Equestrian Affairs, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Farrington, Adrian F
  • Department of Veterinary Clinical Services, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Prabhu, Anil
  • Department of Veterinary Regulation, Welfare and Biosecurity Policy, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Curl, Peter
  • Department of Veterinary Regulation, Welfare and Biosecurity Policy, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Wan, Terence S M
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Ho, Emmie N M
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.

MeSH Terms

  • Anabolic Agents / administration & dosage
  • Anabolic Agents / chemical synthesis
  • Animals
  • Biomarkers / blood
  • Blood Proteins / analysis
  • Chromatography, High Pressure Liquid / veterinary
  • Doping in Sports
  • Horses / blood
  • Male
  • Nitriles / administration & dosage
  • Nitriles / chemical synthesis
  • Orchiectomy
  • Oxadiazoles / administration & dosage
  • Oxadiazoles / chemical synthesis
  • Proteome
  • Proteomics
  • Reproducibility of Results
  • Substance Abuse Detection / veterinary
  • Tandem Mass Spectrometry / veterinary

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Citations

This article has been cited 2 times.
  1. Rooney MF, Neto NGB, Monaghan MG, Hill EW, Porter RK. Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis. Biochem Biophys Rep 2023 Mar;33:101391.
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  2. Peerapen P, Boonmark W, Putpeerawit P, Sassanarakkit S, Thongboonkerd V. Proteomic and computational analyses followed by functional validation of protective effects of trigonelline against calcium oxalate-induced renal cell deteriorations. Comput Struct Biotechnol J 2023;21:5851-5867.
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