NMR-spectra were recorded on a Bruker AvanceTM 600 MHz spectrometer. Mass spectra were conducted with a WATERS LCT Premier XE mass spectrometer (ESI). The UV-Vis absorption spectra were recorded on a Perkin Elmer Lambda 20 UV-Vis spectrometer. Circular dichroism spectra were recorded with a Jasco J-815 spectropolarimeter (Jasco Inc, USA) equipped with the Jasco Peltier-type temperature controller (CDF-426S/15). All measurements were carried out in 1.0 cm path length quartz cells. The infrared spectra were collected, on the diamond crystal surface under vacuum (<1 hPa), using a Bruker Vertex70v FT-IR spectrometer. A Metrohm 902 Titrando digital pH meter, equipped with Tiamo 2.3 software, was used to detect the pH values of the solutions.
Avance 600 mhz spectrometer
Manufactured by Bruker
Sourced in Germany, United States, Switzerland
The Avance 600 MHz spectrometer is a high-performance nuclear magnetic resonance (NMR) instrument produced by Bruker. It operates at a magnetic field strength of 600 MHz, providing high-resolution spectroscopic analysis of samples. The Avance 600 MHz spectrometer is designed for advanced NMR applications in various fields of research and analysis.
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Lab products found in correlation
Sephadex lh 20, by Cytiva (5 mentions)
Topspin, by Bruker (5 mentions)
Topspin 3, by Bruker (5 mentions)
241 polarimeter, by PerkinElmer (4 mentions)
Maxis 4g q tof mass spectrometer, by Bruker (4 mentions)
Equinox 55 s ft ir spectrophotometer, by Bruker (4 mentions)
Avance 400 mhz spectrometer, by Bruker (4 mentions)
Avance 500 mhz spectrometer, by Bruker (4 mentions)
5 mm bbo probe, by Bruker (3 mentions)
Prominence hplc system, by Phenomenex (3 mentions)
Mnova software, by Mestrelab Research (3 mentions)
Avance 400 mhz, by Bruker (3 mentions)
1h 15n 13c triple resonance cryoprobe, by Bruker (3 mentions)
Ls 55 luminescence spectrometer, by PerkinElmer (3 mentions)
Jms 700 mass spectrometer, by JEOL (3 mentions)
Spectrum 100, by PerkinElmer (3 mentions)
J 715 spectropolarimeter, by Jasco (2 mentions)
Uv 1800 spectrophotometer, by Shimadzu (2 mentions)
Rp c18 column, by YMC (2 mentions)
Luna c18 column, by Phenomenex (2 mentions)
185 protocols using avance 600 mhz spectrometer
1
Comprehensive Biophysical Characterization
2
NMR-based Ligand Binding Assay for PD-L1
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NMR experiments were performed using a NMR Bruker AVANCE-TM 600 MHz Spectrometer with a 5 mm BBO probe, the acquisition temperature was set at 25°C. For WaterLOGSY experiments, 0.15 mM of ligand (from a 10 mM stock in DMSO-d6) were added to 5 µM PD-L1 samples in 10 mM sodium phosphate, 25 mM NaCl, ph 7.6 with 10% D2O, in a protein/ligand ratio of 1:30, optimal for the WaterLOGSY experiments. For each compound, samples were prepared with and without protein. For each sample, one-dimensional (1D) 1H and WaterLOGSY experiments were acquired. A total of 16K-points were used for a sweep width of 16 ppm in both experiments. For the 1D 1H experiments, 256 scans were accumulated. A total of 528 scans were accumulated for the WaterLOGSY experiment. Spectra were acquired and processed with TopSpin V.4.1 (Bruker Biospin), and Mnova software (MestReNova, V.14.2.0).
3
Spectroscopic Analysis of Natural Compounds
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NMR spectra were recorded on a Bruker Avance 600 MHz spectrometer. HRESIMS data were obtained using a Xevo G2 Q-TOF mass spectrometer (Waters, Milford, MA, USA). CD spectra were measured on a J-715 spectropolarimeter (JASCO Corporation, Tokyo, Japan). Optical rotations were recorded on a Rudolph IV Autopol automatic polarimeter (Hackettstown, NJ, USA). The UV spectra were recorded on a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). For column chromatography (CC), Sephadex LH-20 (GE Healthcare Bio-Science AB, Pittsburgh, PA, USA), silica gel (200–300 mesh, 300–400 mesh, Tsingtao Marine Chemical Co. Ltd., Tsingtao, China) and RP-C18 (ODS-A, 50 µm, YMC, Kyoto, Japan) were used. Preparative HPLC was run with a P3000 pump (CXTH, Beijing, China) and a UV3000 ultraviolet-visible detector (CXTH, Beijing, China), using a preparative RP-C18 column (5 µm, 20 mm × 250 mm, YMC, Kyoto, Japan). IR data were measured on a Bruker Tensor 27 spectrometer.
4
Preparation and NMR Analysis of Labeled cBAK
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15N-labeled cBAK, produced by calpain proteolysis of MEAS-BAK-ΔTM-His6, as described previously (Moldoveanu et al., 2013 (link)), and 15N-labeled tevBAK were produced in MOPS-based medium (Neidhardt et al., 1974 (link)) supplemented with 15NH4Cl in T7 Express Competent E. coli cells and were purified as described for unlabeled proteins above. Briefly, to produce cBAK (residues 15–186), MEAS-BAK-ΔTM-His6 was batch affinity purified on Ni2+-NTA resin then subjected to S-200 HR size-exclusion chromatography, digestion with μI–II calpain protease for 24–48 h to remove 14 residues at the N-terminus and the C-terminal His6 tag, and MonoQ anion exchange chromatography. 15N-1H TROSY NMR titrations of 50 μM 15N-labeled WT cBAK or R127A(s) C166S G184C tevBAK were performed with fragments dissolved in deuterated DMSO (DMSO-d) at six concentrations (50, 100, 200, 400, 600, and 800 μM), as well as titrations with DMSO-d control at the respective volumes used with the fragments. The NMR buffer contained 20 mM phosphate buffer, pH 6.8, 10% D20, and 2.5 μM deuterated DTT. NMR spectra were obtained with a Bruker Avance 600 MHz spectrometer at 298K, processed in TopSpin (Bruker), and analyzed using CARA (Keller, 2004 ).
5
Metabolic Profiling of Explant Cultures
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Explant culture media samples (200 μL) were collected at D0, D1, and D2 and analyzed by using 1H-NMR spectroscopy (n ≥ 8/condition) according to [61 (link)]. Spectra were accessed at 25 °C by using a Bruker Avance 600 MHz spectrometer with a 5 mm QXI probe and z-gradient (Bruker Biospin, Ettlingen, Germany). Solvent-suppressed 1H-NMR spectra were acquired with 6 kHz spectral width, 14 s inter-pulse, 3 s water pre-saturation, 45-degree pulse angle, 3.5 s acquisition time, and 128 scans (minimum). Sodium fumarate 10 mM (singlet, at 6.50 ppm) was used as an internal reference. The following metabolites were detected and quantified: H1-α glucose (doublet, 5.22 ppm), pyruvate (singlet, 2.35 ppm), alanine (doublet, 1.46 ppm), lactate (doublet, 1.33 ppm), acetate (singlet, 1.9 ppm), and succinate (singlet, 2.39 ppm). The relative areas of 1H-NMR resonances were quantified by using the NUTSproTM NMR spectral analysis program (Acorn NMR, Livermore, CA, USA). D0 media samples were used as the reference/control. Metabolite consumption or production was calculated using the mathematical formula |(D1-D0) + (D2-D0)| [20 (link)] and normalized to the total amount of protein.
6
Comprehensive Analytical Characterization of Compounds
7
Analytical Characterization of DDA
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The resulting product DDA was extracted with methyl tert-butyl ether (MTBE) and dissolved in methanol after complete volatilization of MTBE. Analysis of the product was performed by high-performance liquid chromatography (HPLC) with a UV detector at 210 nm and a reverse phase Nucleosil C18 column (Hypersil ODS2, 4.6 × 250 mm, 5 μm), and the column was eluted with a solvent system of methanol/water/phosphoric acid (95/5/0.1, by volume) at a flow rate of 0.4 mL min− 1 at 30 °C. The retention time of DDA was 7.86 min. 1H-NMR and 13C-NMR measurements were conducted on a Bruker Avance 600 MHz spectrometer.
8
Metabolite Profiling of Cured Ham Samples
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1H NMR spectra of the extracts were recorded on a Bruker Avance 600 MHz Spectrometer (operating at 600.13 MHz, Bremen, Germany) equipped with an ultra-low temperature detection probe at 298 K. The metabolite profile of each ham sample was collected using a standard Bruker pulse sequence NOESYGPPR1D (RD-90-t1-90-tm-90-acquisition) with a weak irradiation during recycle delay (RD, 2 s) and mixing time (tm, 100 ms). A 90° pulse length was set to 15 μs and t1 was adjusted to 2 μs. A total of 32 transients were collected into 32 k data points with a spectral width of 20 ppm. All free induction decays were subjected to an exponential window function with a line broadening factor of 0.3 Hz prior to Fourier transformation. The residual water (δ 4.5~5.0) signal in the spectra was removed. The content of metabolites was measured according to the internal standard (sodium 3-trimethylsilyl propionate). The taste-active values (TAVs) of metabolites were calculated to confirm the taste contribution, according to these descriptions [17 (link),23 (link)].
9
NMR Characterization of Peptides
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Samples were dissolved in 90% H2O/10% D2O (D2O; 99.9% purity; Cambridge Isotope Laboratories, Woburn, MA) with a final concentration of 1 mM at pH 5.5. 4,4-Dimethyl-4-silapentane-1-sulfonic acid (DSS) was used as a chemical shift reference for spectral calibration. One-dimensional (1H) and two-dimensional (TOCSY and NOESY) spectra were recorded for all native and grafted peptides at 298 K on a Bruker Avance 600 MHz spectrometer, with mixing times of 200–300 ms for NOESY experiments. All spectra were assigned using CCPNMR46 (link). All amino acid spin systems were specifically assigned based on Wuthrich et al.47 . For the αH secondary shifts, these were analyzed based on subtracting the random coil 1H NMR chemical shifts of Wishart et al.48 (link) from experimental αH chemical shifts. The three-dimensional molecular structure of SFTI-1 and MCoTI-II were illustrated using MOLMOL49 (link).
10
NMR Characterization of Quadruplexes
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NMR spectra were acquired on a Bruker Avance 600 MHz spectrometer equipped with an inverse 1H/13C/15N/19F quadruple resonance cryoprobehead and z‐field gradients. Quadruplexes were dissolved in a low‐salt buffer with 10 mm potassium phosphate, pH 7.0. For solvent suppression on the samples in 90 % H2O/10 % D2O a WATERGATE with w5 element was employed. Data were processed using Topspin 4.0.6. Proton chemical shifts were referenced relative to TSP.
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