UC Davis

The ability to manipulate the genome and its expression products is of great concern in both human and animal sports due to potential improvements in athletic performance. There are a variety of approaches that have been developed that may allow for alterations of genetic material, including the use antisense oligonucleotides (ASOs) and small interfering RNAs (siRNA), which can interfere with mRNA prior to protein expression. This thesis focused on developing a sensitive, non-targeted Liquid Chromatography – High Resolution Mass Spectrometry (LC-HRMS) method to detect phosphorothioated oligonucleotides in equine serum. Sample preparation involved using solid phase extraction on a mixed mode sorbent, followed by evaporation and concentration steps prior to analysis by LC-HRMS. Extracted oligonucleotides were chromatographically separated using a reverse-phase gradient with ion-pairing reagents prior to introduction to a hybrid quadrupole orbitrap mass spectrometer using negative mode electrospray ionization and all-ion-fragmentation (AIF) and parallel reaction monitoring (PRM) scan modes. The method was validated with percent difference, precision, matrix effects, recovery, limits of detection and quantification, and stability assessed using a representative 13mer synthetic oligonucleotide (PS-1) containing phosphorothioate modifications. The limits of detection (LOD) for the PS-1 oligonucleotide ranged from 10-50 ng/mL, and the limits of quantification (LOQ) ranged from 25-50 ng/mL based on the scan mode with acceptable percent difference and precision. The method was then applied for the detection of two phosphorothioated oligonucleotide sequences targeting either myostatin or EGL9 transcripts that represent gene targets. This LC-MS method successfully detected phosphorothioated oligonucleotides and has potential to be used as a screening method for modified ASOs in equine serum.