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

Cheung, Hiu Wing; Wong, Kin‐Sing; To, Ning Sum; Bond, Amanda J; Farrington, Adrian F.; Prabhu, Anil; Curl, Peter; Wan, Terence S.M.; Ho, Emmie N.M.

doi:10.1002/dta.2988

Cited by 9 publications

(4 citation statements)

References 39 publications

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“…Some suggest the use of metabolomics as an answer, but there are many limitations still to be addressed before this could become an efficient option [ 30 ]. Another possible option would be to test for biomarkers of drug doping, alleviating the need for the time-consuming process of testing for each illicit substance [ [31] , [32] , [33] ]. This would first require identification of such biomarkers.…”

Section: Introductionmentioning

confidence: 99%

Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis

Rooney¹,

Neto²,

Monaghan³

et al. 2023

Biochemistry and Biophysics Reports

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Section: Introductionmentioning

confidence: 99%

Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis

Rooney¹,

Neto²,

Monaghan³

et al. 2023

Biochemistry and Biophysics Reports

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“…To continuously improve their detection capabilities and widen their detection time frames toward major doping agents such as synthetic androgenic anabolic steroids (AAS), doping control laboratories constantly seek analytical enhancements consistent with the processing of several thousand samples per year. These factors limited the application of highly sensitive low-flow based strategies to a few peptide-based targeted applications requiring advanced sensitivity, such as the targeted detection of erythropoietin, , relaxin, peptide toxins, peptide drugs, − or proteomics biomarker discovery induced by doping practices , and unknown protein characterization . To a lower extent, low-flow drug screening methods are reported but are hardly compatible with the processing of tens of thousands samples per year .…”

Section: Introductionmentioning

confidence: 99%

Development of a Standardized Microflow LC Gradient to Enable Sensitive and Long-Term Detection of Synthetic Anabolic-Androgenic Steroids for High-Throughput Doping Controls

Delcourt¹,

Garcia²,

Pottier³

et al. 2021

Anal. Chem.

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Synthetic androgenic anabolic steroids (AAS) are banned compounds and considered as major threats by both racing and sports international authorities. Hence, doping control laboratories are continually looking into analytical improvements to increase their detection capabilities, notably by means of emerging technologies. To enhance analytical performances for the detection of synthetic AAS such as stanozolol, specific chromatographic procedures have been developed using recent quaternary liquid chromatography technology originally designed for highthroughput standardized proteomics connected to mass spectrometry. Applying the newly designed elution procedures described in this paper to the analyses of stanozolol and its metabolites in complex matrixes revealed improved sensitivity compared to previously described high-throughput methods. Indeed, we report the consistent and reliable detection of 16β-hydroxy-stanozolol down to 10 pg/mL in equine urine and being detectable up-to 3 months after a microdosing administration. Furthermore, a five months long elimination of stanozolol and its metabolites could be monitored on horse mane sections after a single dose administration. Our work highlights novel solutions to detect AAS with improved sensitivity. The application of such developments constitutes new landmarks for doping control laboratories and could be extended to other targeted compounds in residue analysis, toxicology, and metabolomics. Based on this work, the developed chromatographic method is now freely available within the Evosep Plus program.

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“…Some suggest the use of metabolomics as an answer, but there are many limitations still to be addressed before this could become an e cient option [30]. Another possible option would be to test for biomarkers of drug doping, alleviating the need for the time-consuming process of testing for each illicit substance [31][32][33]. This would rst require identi cation of such biomarkers.…”

Section: Introductionmentioning

confidence: 99%

Conditionally Immortalised Equine Skeletal Muscle Cell Lines for in Vitro Analysis

Rooney

Neto

Monaghan

et al. 2021

Preprint

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BackgroundThoroughbred racehorse performance is largely influenced by a major quantitative trait locus at the myostatin (MSTN) gene which determines aptitude for certain race distances due to a promoter region insertion mutation influencing functional phenotypes in skeletal muscle. To develop an in vitro system for functional experiments we established three novel equine skeletal muscle cell lines reflecting the variation in phenotype associated with MSTN genotype (CC/II, CT/IN and TT/NN for SNP g.66493737C>T/SINE insertion 227 bp polymorphism). Primary equine skeletal muscle myoblasts, isolated from Thoroughbred horse gluteus medius, were conditionally immortalised and evaluated to determine whether cell phenotype and metabolic function were comparable to functional characteristics previously reported for ex vivo skeletal muscle isolated from Thoroughbred horses with each genotype.ResultsPrimary myoblasts conditionally immortalized with the temperature sensitive SV40TtsA58 lentivirus vector successfully proliferated and could revert to their primary cell phenotype and differentiate into multinucleated myotubes. Skeletal muscle fibre type, MSTN gene expression, mitochondrial abundance, and mitochondrial function of the three MSTN genotype cell lines, were consistent with equivalent characterisation of ex vivo skeletal muscle samples with these genotypes. Furthermore, addition of coenzyme Q10 (CoQ10) to the cell lines improved mitochondrial function, an observation consistent with ex vivo skeletal muscle samples with these genotypes following supplementation with CoQ10 in the diet. ConclusionsThe observation that the phenotypic characteristics and metabolic function of the cells lines are equivalent to ex vivo skeletal muscle indicates that this in vitro system will enable efficient and cost-effective analyses of equine skeletal muscle for a range of different applications including understanding metabolic function, testing of nutritional supplements, drug test development and gene doping test development. In the multi-billion-euro international Thoroughbred horse industry research advances in the biological function of skeletal muscle are likely to have considerable impact. Furthermore, this novel genotype-specific system may be adapted and applied to human biomedicine to improve understanding of the effects of myostatin in human physiology and medicine.

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

Cited by 9 publications

References 39 publications

Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis

Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis

Development of a Standardized Microflow LC Gradient to Enable Sensitive and Long-Term Detection of Synthetic Anabolic-Androgenic Steroids for High-Throughput Doping Controls

Conditionally Immortalised Equine Skeletal Muscle Cell Lines for in Vitro Analysis

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