Sequence determination of phosphorothioated oligonucleotides using MALDI-TOF mass spectrometry for controlling gene doping in equestrian sports.
Abstract: In human and equestrian sporting events, one method of gene doping is the illegal use of therapeutic oligonucleotides to alter gene expression. In this study, we aimed to identify therapeutic oligonucleotides via sequencing using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). As a model of therapeutic oligonucleotides, 22 bp-long phosphorothioated oligonucleotides (PSOs) were used. By using a Clarity OTX kit for extracting short-length oligonucleotides, a spectrum of singly charged PSO with a mean intensity of 6.08 × 10 (standard deviation: 4.34 × 10 ) was detected from 500 pmol PSO in 1 ml horse plasma using the linear negative mode of MALDI-TOF MS. In addition, a 17 bp sequence was determined using in-source decay (ISD) mode, indicating that 500 pmol of a PSO in 1 ml plasma is the detection limit for sequencing. Using the determined sequences (17 bp), a targeted gene for PSO was singly identified on the horse reference genome, EquCab2.0, via a GGGenome search. These procedures can be potentially used to identify therapeutic oligonucleotides, whose nucleotides are unknown, for gene doping control.
© 2021 John Wiley & Sons, Ltd.
Publication Date: 2021-09-08 PubMed ID: 34418319DOI: 10.1002/dta.3154Google Scholar: Lookup
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- Journal Article
Summary
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The research article presents a method to detect the illegal use of therapeutic oligonucleotides in gene doping in equestrian sports by sequencing them using MALDI-TOF mass spectrometry.
Understanding the Context
- Oligonucleotides are short DNA or RNA molecules that can alter gene expression. In the sports world, they could potentially be used illegally to enhance performance – a practice known as gene doping.
- The particular type of therapeutic oligonucleotides that this study focuses on are the 22 bp-long phosphorothioated oligonucleotides (PSOs).
- This research’s goal was to develop a method to identify and sequence these PSO molecules, thus providing a way to detect illegal doping.
The Methodology
- The researchers used 500 pmol of PSOs in 1ml of horse plasma for their study.
- They extracted short-length oligonucleotides using a Clarity OTX kit, an established method for such procedures.
- They then employed matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) for the detection and analysis of the PSOs.
- In MALDI-TOF MS, a laser is used to ionize the molecules (in this case the PSOs), which are then accelerated in an electric field and detected by a time-of-flight detector. The time it takes for the ions to reach the detector is proportional to the mass of the ions, thus providing a way to identify the PSOs.
- They detected PSOs with a mean intensity of 6.08 × 10 (standard deviation of 4.34 × 10).
Findings and Implications
- The researchers were able to determine a 17 bp sequence using in-source decay (ISD) mode, indicating that the detection limit for sequencing is 500 pmol of a PSO in 1 ml plasma.
- They then used the determined sequences (17 bp) to identify a targeted gene for PSO on the horse reference genome, EquCab2.0, using a GGGenome search.
- The successful use of these procedures suggests a viable way to test for the presence of therapeutic oligonucleotides, the sequences of which were previously unknown, adding a new tool for gene doping control methods in equine sports.
Cite This Article
APA
Tozaki T, Kwak HG, Nakamura K, Takasu M, Ishii H, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Hirata M, Nirasawa T, Nagata SI.
(2021).
Sequence determination of phosphorothioated oligonucleotides using MALDI-TOF mass spectrometry for controlling gene doping in equestrian sports.
Drug Test Anal, 14(1), 175-180.
https://doi.org/10.1002/dta.3154 Publication
Researcher Affiliations
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi, Japan.
- Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, Gifu, Gifu, Japan.
- Daltonics Division, Bruker Japan K.K., Yokohama, Kanagawa, Japan.
- Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, Gifu, Gifu, Japan.
- Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, Gifu, Gifu, Japan.
- Drug Analysis Department, Laboratory of Racing Chemistry, Tochigi, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi, Japan.
- Equine Department, Japan Racing Association, Tokyo, Japan.
- Daltonics Division, Bruker Japan K.K., Yokohama, Kanagawa, Japan.
- Daltonics Division, Bruker Japan K.K., Yokohama, Kanagawa, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi, Japan.
MeSH Terms
- Animals
- Doping in Sports / prevention & control
- Gene Expression Regulation / genetics
- Horses / genetics
- Phosphorothioate Oligonucleotides / analysis
- Phosphorothioate Oligonucleotides / blood
- Sequence Analysis
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / veterinary
Grant Funding
- 2020 / Japan Racing Association
References
This article includes 19 references
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Citations
This article has been cited 1 times.- Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Takahashi Y, Nagata SI. Short Insertion and Deletion Discoveries via Whole-Genome Sequencing of 101 Thoroughbred Racehorses.. Genes (Basel) 2023 Mar 3;14(3).
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