Synapt g2 mass spectrometer

All of the fractions obtained in the bio-guided isolation and the pure ACGs were characterized by ¹H-NMR and ¹³C-NMR spectroscopy. ¹H and ¹³C spectra were recorded at room temperature, using CDCl₃ as solvent, on Agilent INOVA spectrometers at 500 and 125 MHz and 600 and 150 MHz, respectively. The residual chloroform signal was set to δ 7.25 ppm for ¹H and to δ 77.0 ppm for ¹³C. Furthermore, ¹H-NMR and ROESYAD experiments on the encapsulated compound were carried out at 25 °C using D₂O as solvent on an Agilent INOVA spectrometer at 500 MHz and 600 MHz. The residual peak for water was referenced to δ 4.79 ppm. HRMS were obtained on a SYNAPT G2 mass spectrometer (Waters, Milford, MA, USA). HPLC was carried out on an HPLC chromatograph with an RI detector (Merck-Hitachi, Tokyo, Japan) and an analytical column Phenomenex^® Luna 10 µ Silica (2) (250 × 4.60 mm, 10 µm) (CA, USA). Silica gel 0.060–0.200, 60 Å from Acros Organics (Geel, Belgium) and Lichroprep RP 18 (40–63 μm) from Merck (Darmstadt, Germany) were used for column and vacuum column chromatography. Thin layer chromatography (TLC) was run on Silica gel 60 F₂₅₄ and Silica gel 60 RP-18 F₂₅₄S aluminum sheets from Merck (Darmstadt, Germany). For further purification, preparative layer chromatography (PLC) Silica gel 60 F₂₅₄ 0.5 mm was used and this was also supplied by Merck (Darmstadt, Germany).

Protein masses were identified using Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) at the Functional Genomics Center Zürich, Switzerland (www.FGCZ.ch) using standard protocols. In brief, samples were diluted 2-fold with 1% trifluoroacetic acid and transferred to autosampler vials for LC/MS. 10 μl of sample was injected into an ACQUITY UPLC@ BioResolve-RP-mAb 2.7μ 2.1x150 450 A (Waters, USA) column. For separation and elution on an Acquity UPLC station (Waters, USA), a gradient buffer A (0.1% formic acid in water)/ buffer B (0.1% formic acid in acetonitrile) at a flow rate of 200 µl/min at 500C over 25 min was applied. Analysis was performed on a Synapt G2 mass spectrometer (Waters, UK) directly coupled with the UPLC station. Mass spectra were acquired in the positive-ion mode by scanning an m/z range from 100 to 4000 Da with a scan duration of 1 s and an interscan delay of 0.1 s. The spray voltage was set to 3 kV, the cone voltage to 50 V, and source temperature 80 °C. The data were recorded with the MassLynx 4.2 Software (Waters, UK). For single peaks, the recorded m/z data were then deconvoluted into mass spectra by applying the maximum entropy algorithm MaxEnt1 (MaxLynx) with a resolution of the output mass 0.5 Da/channel and Uniform Gaussian Damage Model at the half height of 0.5 Da.

YdaT was buffer-exchanged into 200 mM ammonium acetate pH 8 using Amicon Ultra 0.5 ml centrifugal filters (Merck Millipore) with a molecular-weight cutoff of 3 kDa. The concentrations of protein and oligonucleotide alone were 2.5 µM (tetramer concentration) and 5 µM (DNA duplex), respectively. The complexes between YdaT and oligonucleotide were prepared at different YdaT tetramer:DNA molar ratios (0.25:1, 0.25:1.5, 0.5:1, 0.75:1, 1:1, 1.25:1 and 1.5:1), keeping the protein concentration fixed at 2.5 µM YdaT tetramer. Native mass spectrometry was performed on a Synapt G2 mass spectrometer (Waters). The samples were introduced into the gas phase through nano-electrospray ionization with in-house-prepared gold-coated borosilicate glass capillaries. The settings were optimized for the analysis of larger structures as natively as possible. The critical voltages and pressures used were a sampling cone voltage of 50 V and a trap collision energy of 10 V, with pressures throughout the instrument of 6.18 and 2.42 × 10⁻² mbar for the source and trap collision cell regions, respectively. Analysis of the acquired spectra was performed using MassLynx version 4.1 (Waters). Native MS spectra were smoothed (to an extent depending on the size of the complexes) and additionally centred to calculate the molecular weights to determine precise stoichiometries.