PDF(Pubmed)
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.
摘要：
在马运动中，人们非常担心能够编辑基因组或修饰基因产物表达的药物的潜在用途。合成的寡核苷酸很短，单链多核苷酸,其代表一类能够修饰基因表达产物的试剂,其在赛马中具有高的滥用潜力。由于大多数常规反兴奋剂分析方法都不包括这些物质，它们代表不容易检测到的化合物的整个类别。每个寡核苷酸的核苷酸序列是高度特异性的，这使得对这些药物的针对性分析存在问题。因此，我们开发了一种非靶向方法来检测核糖核酸中天然不存在的特定产物离子的存在。简而言之，在破坏蛋白质相互作用以分离寡核苷酸后，使用混合模式盒进行固相萃取提取血清样品。在洗脱和浓缩步骤之后，色谱分离是利用反相液相色谱法实现的。在介绍了使用电喷雾电离的ThermoQExactiveHF质谱仪之后，使用全扫描组合检测分析物，并行反应监测和所有离子碎片扫描模式。检测限与准确性一起确定，精度,稳定性,recovery,和使用代表性13mer寡核苷酸的基体效应。在使用13mer寡核苷酸进行方法优化之后，该方法被用于成功地检测在三个独特的寡核苷酸序列中特定产物离子的存在针对马特异性转录本。
