6230 tof mass spectrometer

Flexizyme activity was assayed as follows. Two microliters of 7.5 µM chimeric or all-RNA flexizyme was treated with 200 mM NaOH for 30 s at room temperature before being quenched with equimolar HCl (final volume of the quenched reactions was 3 µL). In parallel, 7.5 µM chimeric or all-RNA flexizyme was diluted with water to 5 µM and incubated for 30 s at room temperature in triplicate. The reactions were diluted to 2.5 µM flexizyme with 50 mM Na⁺-HEPES, pH 8.0, 5 mM Gly-DBE (20% DMSO), 10 µM substrate oligonucleotide, and 10 mM MgCl₂ and incubated at 0 °C. Reaction aliquots (0.5 µL) were quenched in 4.5 µL of acidic quenching buffer (10 mM EDTA, 100 mM NaOAc, pH 5.0, 150 mM HCl, and 70% [vol/vol] formamide) at the indicated time points and resolved on a 20% acidic polyacrylamide gel (19:1 with 7 M urea and 0.1 M NaOAc, pH 5.0). The gel was run for 2 h at 300 V at 4 °C and visualized with a Typhoon 9410 imager. Aminoacylation percentages were quantified by measuring band densities in ImageQuant TL 8.1 software. The remaining aminoacylation reactions at 22 h were desalted using Zip-Tip C18 columns and characterized by high-resolution mass spectrometry on an Agilent 6230 TOF mass spectrometer.

HPLC-MS analysis was performed using an Agilent 1200 Infinity LC coupled to an Agilent 6230 TOF mass spectrometer with an Agilent Dual ESI Source (ESI SG14289023) and MassHunter Workstation Software, version B.05.01, Build 5.01.5125.3, operating in negative ionization mode. The capillary voltage was 4500 V; the dry gas temperature was 300 °C; nitrogen was used as the dry gas at a flow rate of 7 L/min; the nebulizer pressure was 60 psig; the fragmentor was 200 V; the MS range was 50–1500 m/z; and the MS acquisition rate was 1 spectrum/s. The chromatographic separation was accomplished using an HPLC system (Infinity Series 1200, Agilent Technologies, Waldbronn, Germany) equipped with a Kinetex 2.6 u, C1800A column (150 × 2.1 mm) (Phenomenex, SA, Torrance, CA, USA). The column temperature was maintained at 25 °C. The following gradient program was used along with a mobile phase consisting of water: acetonitrile (90:10) with 1.0% formic acid (solvent A) and methanol:acetonitrile (90:10) with 1.0% formic acid (solvent B). These initial conditions were 3 min in an isocratic elution composed of 100% solvent A followed by 3–5 min: 65% A-35% B; 5–15 min: 50% A-50% B; 15–30 min: 100% B; and 40 min: 100% B, v/v. The flow rate was 0.15 mL/min, and the injection volume was 6 μL (0.1 mg/mL) for the mangiferin standard and 10 μL (0.3 mg/mL) for methanolic extract.

Oligonucleotides were either
purchased from Integrated DNA Technologies (Coralville, IA) or synthesized
in house on an Expedite 8909 solid-phase oligo synthesizer. Phosphoramidites
and reagents for the Expedite synthesizer were purchased from either
Glen Research (Sterling, VA) or Chemgenes (Wilmington, MA). Cleavage
of synthesized oligonucleotides from the solid support was performed
using 1 mL of AMA (1:1 mixture of 28% aqueous ammonium hydroxide and
40% aqueous methylamine) for 30 min at room temperature, while deprotection
was performed in the same solution for 20 min at 65 °C. Deprotected
oligos were lyophilized, resuspended in 100 μL of DMSO and 125
μL of TEA·3HF, and heated at 65 °C for 2.5 h to remove
TBDMS from 2′-hydroxyls. Following this deprotection, oligos
were purified by preparative 20% polyacrylamide gel electrophoresis
(19:1 with 7 M urea), desalted using Waters (Milford, MA) Sep-Pak
C18 cartridges, and characterized by high-resolution mass spectrometry
on an Agilent 6230 TOF mass spectrometer.

Agilent 6230 TOF mass spectrometer was used to determine the concentration of the known compounds from the CKI and reconstituted CKI-OmtOspc and CKI-MacOmtOspc mixtures. 10 µl sample was injected with a flow rate of 0.8 ml/min, a gradient program of 0 min, 100% A; 25 min, 40% B; 35 min, 60% B; and solvents MilliQ H₂O + 0.1% formic acid (solvent A) and acetonitrile + 0.1% formic acid (solvent B). The column used was C₁₈ (5 μm, 150 × 4.6 mm, Diamosnsil, Dkimatech, China). The recovered contents of the samples were measured by spike-in compound cytosine. Gas phase ions were generated with an electrospray source, with key instrument parameters: gas temperature, 325; sheath gas temperature, 350; vCap, 3500; fragmentor, 175; acquisition range (m/z) 60–17000. Calibration curves for nine standard compounds containing various concentrations were shown in Supplementary Data.

LC–MS analysis was performed using an Agilent 1260 LC stack equipped with a Zorbax RX-C18 column (5 μm, 4.6 × 150 mm) and connected to an Agilent 6230 TOF mass spectrometer with electrospray ionization (ESI). Analyses used an isocratic mixture containing 65% water, 25% acetonitrile, and 10% isopropanol at a flow rate of 0.5 mL/min. The mass spectrometer was run in the negative ion mode with a probe voltage of 4,500 V and a fragmentation voltage of 175 V. To monitor NNG, 2-NAE, and glyoxylate, extracted ion chromatograms were obtained at m/z 119.0, 105.0, and 73.0, respectively.
Ammonium concentrations were determined using a glutamate dehydrogenase assay (Sigma-Aldrich) kit using the manufacturer’s instructions. Nitrite concentrations were determined by reacting 25 μL aliquots of reaction sample with 25 μL of deoxygenated Griess reagent R1 (1% sulfanilamide in 5% H₃PO₄) followed by addition of 25 μL of deoxygenated Griess reagent R2 (0.1% naphthylethylenediamine dihydrochloride in water). The absorbance was read at 548 nm using an Infinite M200 Plate Reader (Tecan). Nitrite concentrations were determined by comparison of A_{548 nm} to a nitrite standard curve.