Prodigy tci cryoprobe

NMR spectra were recorded at 600 MHz on a Bruker Avance III spectrometer, equipped with a TCI CryoProbe or on Bruker Avance NEO spectrometer equipped with a Cryo probe Prodigy TCI. The spectra were recorded at 25 °C. Typical ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectra were performed with a data matrix consisting of 2048 (F2, ¹H) × 256 (F1, ¹⁵N) complex points, spectral windows of 8417.509 Hz (¹H) × 2432.717 Hz (¹⁵N), 16 transients, and 1.2 s relaxation delay. Unless otherwise specified, 2D experiments were run on samples at a protein concentration of 100 µM with 2 mM Ca²⁺ and with 2 mM Ca²⁺ and 200 µM RyR2 peptide, dissolved in 20 mM TRIS pH 7.5, 150 mM KCl, 7% D₂O buffer.
NMR titration experiments were run on samples with a protein concentration of 300 µM in 20 mM TRIS, pH 7.5, 150 mM KCl, 0.4 mM EGTA, 7% D₂O buffer with Ca²⁺ ions added stepwise from a concentrated stock solution (100 mM). The following protein/ligand rations were analyzed by SOFAST-HMQC (Band-Selective Optimized Flip Angle Short Transient) spectra: 1:0, 1:0.5, 1:1.5, 1:7, 1:20. Each experiment was performed with a data matrix of 960 (F2, ¹H) × 160 (F1, ¹⁵N) complex points, spectral windows of 8196.722 Hz (¹H) × 1824.533 Hz (¹⁵N), 8 transients, and 0.1 s relaxation delay. All data were processed and analyzed using TOPSPIN 4.1.1 (Bruker, Karlsruhe, Germany).

The detailed measurement protocol has been previously published [3 (link)]. Shortly, the urine NMR data were measured with Bruker AVANCE III HD (Bruker BioSpin GmbH, Karlsruhe, Germany) 600 MHz spectrometer equipped with a Bruker Prodigy TCI cryoprobe and an automatic cooled SampleJet sample changer. Standard water suppressed spectra (noesygppr1d) were measured using the following parameters: number of scans 16, spectral width 21.0297 ppm, size of fid 81,920, acquisition time 3.2440 s, relaxation delay 2.5 s, and receiver gain 57. The spectra were measured at 295 K. The spectra were processed and phase-corrected in an automated fashion. The free induction decays were zero-filled to 128 k data points and multiplied with an exponential window function with a 0.3 Hz line broadening.

Proton nuclear magnetic resonance spectroscopy (¹H NMR) metabolomics analysis was performed from plasma by following the protocol described previously [18 (link)]. The samples were prepared and analysed in a randomized order. Briefly, an aliquot of 220 µL plasma was mixed with 440 µL phosphate buffer (90 mmol/L NaH2PO4, pH = 7.4) containing 15% D₂O. After centrifuging, 600 µL of the resulting supernatants were transferred into 5-mm NMR tubes. The NMR experiments were performed at 298 K on a 600 MHz Bruker Avance-III NMR spectrometer (Bruker BioSpin AG, Fällanden, Switzerland) equipped with a Prodigy TCI cryoprobe and a precooled SampleJet sample changer. Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence was used. Metabolites were identified based on 1D CPMG NMR chemical shifts reported in Chenomx NMR Suite 7.5 software (Chenomx Inc., Edmonton, AB, Canada), 2D NMR (¹H−¹³C heteronuclear single-quantum correlation spectroscopy (HSQC) and ¹H−¹H correlation spectroscopy (COSY)), and the metabolite database Human Metabolome Database (HMDB, http://www.hmdb.ca, accessed on 16 August 2022). The Representative CPMG spectrum of plasma samples is shown in Figure S1. Altogether, 22 metabolites were identified using Chenomx and 2D NMR (Figures S2 and S3), and their chemical shifts and peak multiplicity are summarized in Table S1.