Xevo g2 xs

The Covid‐19 Spike Protein aptamer was synthesized under standard conditions at 1 µmol scale using modified polymerizable T bases (5′‐**T***T********T***T*******T***T*‐3) on an Applied Biosystems 394 oligonucleotide synthesizer. The polymerizable base used in this study was carboxy‐dT, a thymine modified with a carboxyvinyl moiety on the 5′ position. This was used in previous studies and has demonstrated excellent incorporation in the polymer matrix.^[23, ²⁴, ^26] The synthesized oligomers were deprotected and released from the support by treatment with concentrated aqueous ammonia at 60 °C for 24 h. The solutions were concentrated to dryness, resuspended in water, and purified by semi preparative HPLC on an Agilent 1260 infinity system with a Phenomenex Clarity 5 µm Oligo‐RP LC 250 × 10 mm column. Collected fractions were desalted using NAP‐10 columns (GE Healthcare) and oligo purity was determined by analytical HPLC on an Agilent 1260 infinity system with a Phenomenex Clarity 5 µm Oligo RP LC 250 × 4.6 mm column. Oligonucleotide masses were verified using a Waters Xevo G2‐XS, and concentrations were determined by optical density at 260 nm using a BioSpec‐nano micro‐volume UV‐Vis spectrophotometer (nanodrop, Shimadzu), and the Beer Lambert law, with extinction coefficients obtained from OligoAnalyzer (Integrated DNA Technologies).

To obtain measurements of microcystin concentrations, 5 mL was withdrawn from each flask at each time point and stored in 15 mL Corning® polypropylene centrifuge tubes at −80 °C for later analysis. Microcystins were analyzed using on-line solid phase extraction coupled to liquid chromatography-quadrupole time-of-flight high resolution mass spectrometry (Waters Xevo G2-XS, Milford, MA, USA), optimized by Ortiz et al. [31 ]. In this targeted and non-targeted high throughput method, samples of M. aeruginosa CPCC 300 at each time point were measured and 12 microcystin variants (LR, YR, RR, HtyR, HilR, WR, LW, LA, LF, LY, Dha₇-LR, and Dha₇-RR) and anatoxin-A (all standards purchased from Enzo Life Sciences, Farmingdale, NY, USA) were quantified using nodularin as the internal standard. Using this high throughput method, batches of 50 samples can be prepared for instrumental analysis in less than 3 h. Detection limits were 0.05 μg L⁻¹ with an expanded uncertainty ranging from 4 to 14 % for the different variants, which takes into account the uncertainty coming from the sample preparation, the instrument, and calibration standards. The microcystin cell quota (Q_mcyst), expressed as femtograms (fg) microcystin per cell, was calculated by dividing the concentration of each detectable microcystin congener (fg mL⁻¹) by the cell concentration (cells mL⁻¹) (Fig. 1).

LC-MS was performed using an ultrahigh-performance liquid chromatography (UPLC) system (H-class, Waters, USA) coupled with a quadrupole time-of-flight tandem spectrometer (Xevo G2-XS, Waters, USA). Separation was performed using a Waters Acquity UPLC BEH C18 column (3.0 mm × 100 mm, 1.7 μm, Waters, USA). The column temperature was 30°C. The detection wavelength was set at 196 nm for all the tested compounds, and the mobile phases were acetonitrile (A) and water (B). A gradient elution was used: 0 min, 20% (v/v) A; 20 min, 50% (v/v) A; 30 min, 20% (v/v) A. The mobile phase was established at a flow rate of 0.2 mL·min⁻¹, and the injection volume was 5 μL.
The nebulization gas was set to 600 L·h⁻¹ at a temperature of 350°C, and the cone gas was set to 50 L·h⁻¹. The source temperature was set to 110°C. The capillary voltage and cone voltage were set to 3500 V and 30 V, respectively. The data acquisition rate was set to 0.3 s with a 0.1 s interscan delay. Data between m/z 50 and 1200 were recorded in positive ion mode. The quality axis was corrected by sodium formate, and the quality of leucine enkephalin was corrected in real time.

The analyses were performed using a Waters Xevo G2-XS quadrupole-time
of flight mass spectrometer for compound characterization in HR (Supporting
Information, section “Supplementary Results,” MS And MS/MS Characterization) and a Waters Xevo TQ-XS
(triple quadrupole) mass spectrometer both equipped with Z-spray (electrospray)
ionization and step-wave source optimization, and controlled under
MassLynx v4.2 (Waters) as fully described previously.^{13 (link)} The Waters Xevo G2-XS was used for the compounds characterization
in HR, in full-scan, and in quadrupole selection mode without or with
increasing collision energy up to 35 V for fragment generation. The
Waters Xevo TQ-XS was used for the implementation of the MRM method
as detailed in Supporting Information, section “Supplementary Results” and Table S1. The final
MRM transitions for O6-m2dGO and 2dGO analyses are given in Table 2 with expected LC
retention times of each compound and selected MRM transitions for
LC method optimization.
MassLynx and TargetLynx v.4.2 (Waters) were used for
rapid chromatogram
and spectra evaluation and for the construction of calibration curves
and the computing of quantification data, respectively. As only 60
μL out of 65 μL was taken from each sample for analysis
and CALs were prepared as 60 μL-samples, calculated concentrations
were corrected by a factor 1.083 to retrieve real concentrations in
the samples.

Frozen samples (0.15–0.17g) were grinded and phytohormones were extracted with 1ml of ethyl acetate (Lee et al., 2015 (link)). Ultra-performance liquid chromatography (ACQUITY®UPLC system, Waters Corp., Milford, MA, USA) coupled with a QTOF instrument (XEVO G2XS; Waters Corp.) was used for the analysis. The chromatographic separation was performed on an ACQUITY®UPLC BEH C18 column (100×2.1mm, i.d. 1.7 μm). The mobile phases consisted of solvent A (0.1% formic acid) and solvent B (acetonitrile). The gradient elution mode was programmed as follows: 20–25% B for 0.0–5.0min and 25–35% B for 5.0–10.0min. Mass spectrometry analysis was conducted in the negative ion mode with electrospray ionization (ESI).

Ultra-performance liquid chromatography (UPLC) analysis was performed using an ACQUITYVRUPLC system (Waters Corp., Milford, MA, United States) coupled to a Q-TOF instrument (XEVO G2XS; Waters Corp.). The chromatographic separation was carried out on an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, i.d., 1.7 μm) connected to an ACQUITY UPLC BEH C18 VanGuard pre-column (5 mm × 2.1 mm, i.d., 1.7 μm). The mobile phases consisted of solvent A (0.1% formic acid) and solvent B (acetonitrile). The gradient elution mode was programmed as follow: 5–60% B for 0.0–7.5 min and 60–95% B for 7.5–10.0 min. The column was then washed with 95% B for 3 min and equilibrated with 5% B for 2 min. All samples were kept at 10°C during the analysis. The flow rate and injection volume were 0.4 mL/min and 2 μL, respectively. MS analysis was conducted in the negative ion mode with electrospray ionization (ESI). The MS conditions were optimized as follows; capillary voltage, 3 kV; cone voltage, 40 V; source temperature, 130°C; desolvation temperature, 400°C; cone gas flow, 50 L/h; desolvation gas flow, 900 L/h.