52 August / September 2019
deproteinated by adding 30 mL acetonitrile (LC/MS grade, Wako, Osaka, Japan) to 15 mL horse plasma in 50 mL tubes, then mixing vigorously, storing at 4°C for 15 min, centrifuging at 3000xg for 15 min, and collecting 40 mL of the supernatant. Remaining solvent was removed using an evaporator, and the final volume was adjusted to 10 mL with Milli-Q water. The deproteinated horse plasma samples were then stored at –20°C until use.
For the PSO, a 22-mer antisense sequence of exon 2 of the equine myostatin gene (MSTN), 5ʹ-GAG ATC GGA TTC CAG TAT ACCA-3ʹ, was synthesised, with all the nucleotides phosphorothioated (GeneDesign,Inc. Osaka, Japan). The PSO was purified using HPLC, then dissolved in 100 mmol/L triethylamine acetate (TEAA) buffer at a concentration of 20 µg/mL. One set of the equine plasma samples were then spiked with PSO to concentrations ranging from 1 ng/mL to 20,000 ng/mL. To evaluate any false positives, unphosphorothioated 15-mer oligo dTs, 5ʹ-TTT TTT TTT TTT TTT-3ʹ (GeneDesign, Inc.), were spiked into all the test and blank plasma samples to a concentration of 4.5 µg/mL.
High performance liquid chromatography
The blank and test equine plasma samples were analysed first with HPLC on a Prominence UFLC XR HPLC system (Shimadzu, Kyoto, Japan) using an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 × 30 mm, Waters, Milford, MA, USA). The column was maintained at 60°C, with a mobile phase consisting of Solvent A: 100 mM hexafluoro- 2-propanol [HFIP] and 10 mM triethylamine (TEA), and Solvent B: methanol. The sample injected in 2 µL volumes, with the elution gradient as: 5 to 50 to 90% Solvent B in 2 and 0.01 min, then hold for 0.49 min, then 0.01 min in 5% Solvent B, then hold for 1.49
Table 1: Accuracy and reproducibility of PSO detection. Sample Type Concentration (ng/mL) Standard
0.1 0.2 0.5 1.0 2.0 5.0
10.0 2.0
Quality Control
100.0 200.0 20.0
200.0 Accuracy (%) Day 1
103.0 103.5 82.5 89.8 91.6 98.7
113.1 114.7 104.0 97.4
111.5 96.1
Day 2
108.5 93.4 82.0 86.6 94.2 90.9
117.4 117.2 109.8 95.4
119.3 102.4
min, after pre-equilibrium for 0.5 min in 5% Solvent B at a flow rate of 0.4 mL/min.
Targeted and non-targeted spectroscopic analyses to selectively detect PSOs in equine plasma
After HPLC separation, the plasm samples were analysed with quadrupole-time-of- flight (Q-TOF) tandem mass spectroscopy (MS/MS) on a TripleTOF® 6600 System (SCIEX, Framingham, MA, USA) equipped with a DuoSpray™ ion source.
For targeted analysis, a product ion scan was performed for the precursor ion of mass-to- cha rge ration (m/z) of 785.18 corresponding to [M−9H]9−
, and the Q1 resolution was
set to low to transmit the isotope ions and increase the sensitivity. Then, the product ion of m/z 94.9362 derived from the PS moiety was detected using high-resolution multiple reaction monitoring (MRMHR
70–2800 for the product ion scan.
The data acquisition was information- or data-dependent (IDA or DDA). Dynamic background subtraction (DBS) was applied to the acquisition to minimise the collection of MS/MS spectra on background ions, thus increasing the identification of low-abundance analytes in the presence of background noise. Data acquisition, targeted analysis, and non-targeted analysis were performed using Analyst®TF 1.7.1, MultiQuant™ 3.0.2, and PeakView® 2.2 software (SCIEX), respectively.
Results and Discussion
Targeted and non-targeted spectroscopic analyses selectively detected PSOs in equine plasma
) in high
sensitivity and enhanced ion modes, which provided additional ion selectivity. The MS parameters used were a scan range of m/z 70–300, Gas 1 (nebuliser gas) at 60 psi, Gas 2 (heater gas) at 80 psi, curtain gas at 25 psi, at an ion source temperature of 600°C, an ion spray voltage of − 4000 V, a declustering potential of − 120 V, and collision energy (CE) of − 92 V. The mass spectrometer was calibrated with external standard solution before acquisition.
For non-targeted analysis, information- or data-dependent acquisition (IDA or DDA) was used to search for the PSOs that could produce the specific product ion of negatively charged PS. The product ion scan data was acquired by triggering the 10 most intense precursor ions within the DDA criteria every 0.2 s cycle. The same MS parameters were used as for the targeted analysis, except the scan ranges, which were m/z 450–2800 for the full scan and m/z
In the targeted spectroscopic analysis, the MRMHR
enhanced ion mode enabled
the target product ions to reach the detector 16 times more efficiently. The target analyte was detected with a limit of detection (LOD) of 0.1 ng/mL. In quantitative analysis, the calibration curves generated using quadratic regression demonstrated excellent linearity in the range of 0.1–200 ng/mL with a correlation coefficient (R) of ≥ 0.990 (see Figure 2). Accuracy measures of 82–118% were observed and intra- and inter-day reproducibility were confirmed (see Table 1). The sensitivity of the quantitation also indicates that the product ion of m/z 94.9362 could serve as a good marker for pharmacokinetic studies of PSOs.
In the non-targeted spectroscopic analysis, the specific product ion of m/z 94.9362 was detected in the PSO-spiked plasma and not in the blank samples (see Figure 3). Therefore, the detected product ion of m/z 94.9362 did not originate from the endogenous components of the plasma. The PSO analyte was detected at a concentration of 100 ng/mL at a retention of approximately 1.4 minutes. No interfering peaks were detected at or around 1.4 minutes in the blank samples. Moreover, when filtered to the product ion of m/z 94.936, peaks were detected in the spiked samples, while no peaks were detected in the blanks (see Figure 4). Phosphorothioated oligomers were thus selectively detected in a heavy biological matrix containing a 45 times higher concentration of a non-PS oligomer without any sequence information.
A marker-based test proposed for the detection of gene doping These findings indicate that the product ion
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