Non-Targeted Detection of Synthetic Oligonucleotides in Equine Serum Using Liquid Chromatography-High-Resolution Mass Spectrometry.
Abstract: There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression products with a high potential for abuse in horseracing. As these substances are not covered by most routine anti-doping analytical approaches, they represent an entire class of compounds that are not readily detectable. The nucleotide sequence for each oligonucleotide is highly specific, which makes targeted analysis for these agents problematic. Accordingly, we have developed a non-targeted approach to detect the presence of specific product ions that are not naturally present in ribonucleic acids. Briefly, serum samples were extracted using solid-phase extraction with a mixed-mode cartridge following the disruption of protein interactions to isolate the oligonucleotides. Following the elution and concentration steps, chromatographic separation was achieved utilizing reversed-phase liquid chromatography. Following an introduction to a Thermo Q Exactive HF mass spectrometer using electrospray ionization, analytes were detected utilizing a combination of full-scan, parallel reaction monitoring and all ion fragmentation scan modes. The limits of detection were determined along with the accuracy, precision, stability, recovery, and matrix effects using a representative 13mer oligonucleotide. Following method optimization using the 13mer oligonucleotide, the method was applied to successfully detect the presence of specific product ions in three unique oligonucleotide sequences targeting equine-specific transcripts.
Publication Date: 2024-05-25 PubMed ID: 38891955PubMed Central: PMC11172053DOI: 10.3390/ijms25115752Google Scholar: Lookup
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- Journal Article
Summary
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The research has successfully developed a non-targeted method to detect the presence of synthetic oligonucleotides, known for modifying gene expressions, in equine serum. This method was created due to worries about these substances being used for unfair advantages in horse racing.
Understanding the Research Problem
- The research is set against the backdrop of ethical concerns in equine sport where prohibited pharmaceutical agents are potentially being used to enhance performance. By modifying the genome or altering gene product expressions, synthetic oligonucleotides could be abused in this context.
- The trouble is that most routine anti-doping procedures do not cover these substances, therefore they are difficult to detect.
- Complicating the problem is the high specificity of each oligonucleotide’s nucleotide sequence, making targeted analysis of these agents a challenge.
The Non-Targeted Approach
- In light of these concerns, the researchers develop a non-targeted approach providing a way to detect the presence of certain product ions that do not naturally occur in ribonucleic acids.
- The process involves extracting serum samples via solid-phase extraction using a mixed-mode cartridge. This is done following the disruption of protein interactions with the purpose to isolate the oligonucleotides.
- Chromatographic separation was then carried out using reversed-phase liquid chromatography after the elution and concentration steps.
Use of Mass Spectrometry
- The extracted samples were introduced to a Thermo Q Exactive HF mass spectrometer using electrospray ionization. Here, analytes were detected using a combination of full-scan, parallel reaction monitoring, and all ion fragmentation scan modes.
- Still, important factors such as the limits of detection, accuracy, precision, stability, recovery, and matrix effects were determined using a representative 13mer oligonucleotide.
- After this method had been optimized using the 13mer oligonucleotide, this method was applied and successfully able to detect the presence of specific product ions in three unique oligonucleotide sequences targeting equine-specific transcripts.
Conclusion
- This research offers a promising solution in the detection of synthetic oligonucleotides that can be used to modify gene expressions in horses, something undetectable with routine anti-doping tests.
- The researchers’ non-targeted approach using reversed-phase liquid chromatography and a Thermo Q Exactive HF mass spectrometer can aid in maintaining ethics in equine sports, particularly horse racing.
Cite This Article
APA
Helmes E, Montgomery J, Alarcio G, Mendoza HG, Blea JA, Beal PA, Moeller BC.
(2024).
Non-Targeted Detection of Synthetic Oligonucleotides in Equine Serum Using Liquid Chromatography-High-Resolution Mass Spectrometry.
Int J Mol Sci, 25(11), 5752.
https://doi.org/10.3390/ijms25115752 Publication
Researcher Affiliations
- KL Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
- KL Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
- KL Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
- Department of Chemistry, University of California, Davis, CA 95616, USA.
- School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
- Department of Chemistry, University of California, Davis, CA 95616, USA.
- KL Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
MeSH Terms
- Animals
- Horses / blood
- Oligonucleotides / blood
- Doping in Sports / prevention & control
- Chromatography, Liquid / methods
- Mass Spectrometry / methods
- Solid Phase Extraction / methods
- Limit of Detection
Grant Funding
- Parimutual revenue / California Horse Racing Board
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
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