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29 protocols using avance drx 600

1

HR MAS MRS of Tumor Biopsies

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To minimize the effect of tissue degradation on the metabolite profiles, the samples were prepared on ice block and within a short period (5 ± 1 min). The biopsies (13 ± 3 mg) were cut to fit 30 μl disposable inserts filled with 3 μl phosphate buffered saline (PBS) in D2O containing 1.0 mM TSP for chemical shift referencing and 1.0 mM Format for shimming. The HR MAS spectra were acquired on a Bruker Avance DRX600 spectrometer equipped with a 1H/13C MAS probe with gradient (Bruker Biospin GmbH, Germany) using the following parameters; 5 kHz spin rate, 4°C probe temperature, cpmgpr1D sequence (Bruker Biospin GmbH, Germany) with 273.5 ms total echo time, a spectral width of 20 ppm (−5 to 15 ppm) and 256 scans (NS). For some patients, more than one biopsy (taken from different places in the tumor) were prepared and analyzed by HR MAS MRS.
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2

NMR Experiments for Protein Resonance Assignment

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NMR experiments were conducted at 25 °C on Bruker Avance III HD (600 and 700 MHz), Bruker Avance/DRX 600 (600 MHz) and Varian VNMRS (900 MHz) instruments, equipped with cryogenic Z-axis pulse-field-gradient (PFG) triple resonance probes. For 1H and 13C chemical shifts 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) was used as internal standard at 0 ppm. 15N chemical shifts were referenced indirectly using frequency ratios.44 (link) For NMR kinetics at Bruker Avance/DRX 600, 1H measurements were recorded relative to the resonance of the solvent. Assignment of protein backbone resonances was performed using a combination of multidimensional NMR experiments: 2D [1H–15N]-TROSY,45 (link) 3D HNCA,46 (link) 3D HN(CO)CA,47 (link) 3D HNCO,48 3D HN(CA)CO49 and 3D [1H–1H–15N]-TOCSY-HSQC22 (link) experiments at 900 MHz. Raw data for backbone assignment were processed with NMRPipe v.8.1 (ref. 50 ) and analyzed with CppNmr v.2.4.51
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3

HR MAS 1H NMR Spectroscopy for Ocular Tissues

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HR MAS 1H NMR (high-resolution magic angle spinning proton nuclear magnetic resonance) spectroscopy was performed on a Bruker Avance DRX600 spectrometer (14.1 T, Bruker BioSpin GmbH, Rheinstetten, Germany) operating at 600.132 MHz for protons. Tissue samples were immersed in D2O in a zirconia 4 mm diameter (50 μL) HR-MAS rotor. Sodium [2,2,3,3-d4]3′-trimethylsilylpropionate (d4-TSP, 25 mM) was used as an internal shift reference. The spectra were recorded at 4°C using a 4 mm 1H/13C MAS probe. The samples were spun at 5000 Hz and the number of scans was 512. Water suppression was done employing a presaturation selective pulse. Exponential line broadening of 0.3 Hz was used. Carr-Purcell-Meiboom-Gill (CPMG) spectra analysis was conducted using special software for the analysis of complex mixtures (MestreNova 5.1.0, Mestrelab Research, Spain). Peak areas were measured using absolute integrals and were normalised by the wet weight of the samples (weight range of corneas: 7.3–12.3 mg; ciliary bodies: 7.5–12.4 mg; iris: 7.6–12.1 mg; lens: 7.4–12.5 mg; retinas: 7.2–12.3 mg) and assignment of the metabolites in the spectra were performed as described earlier [4 (link)].
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4

NMR Spectroscopy of Biological Samples

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Samples were resuspended in 0.6 ml NMR buffer consisting of 0.1 M phosphate buffer pH 7.4, 1 mM trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (TSP), and 0.55 ml was transferred to a 5 mm NMR tube. Spectra were acquired on a Bruker Avance DRX600 NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany), with 1H frequency of 600 MHz, and a 5 mm inverse probe. Samples were introduced with an automatic sampler and spectra were acquired following the procedure described by Beckonert et al [15 (link)]. Briefly, a one- dimensional NOESY sequence was used for water suppression; data were acquired into 64 K data points over a spectral width of 12 KHz, with 8 dummy scans and 512 scans per sample. In addition, 2D 1H-1H COSY were acquired. Data were acquired into 512 × 4096 data points covering 6 × 6 KHz, with 3 scans for each increment.
Spectra were processed in iNMR 2.6.3 (Nucleomatica, Molfetta, Italy). Fourier transform of the free-induction decay was applied with a line broadening of 0.5 Hz. Spectra were manually phased and automated first order baseline correction was applied. Metabolite assignments were based on Tredwell et al [14 (link)]. Metabolite concentrations from 2D spectra, relative to the internal standard TSP were calculated using rNMR [16 (link)], and data were normalised by the probabilistic quotient normalisation method described by Dieterle et al [17 (link)].
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5

Analytical Techniques for Compound Characterization

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Ultraviolet (UV) spectra were measured on a Hitachi 300 Spectrophotometer. Nuclear magnetic resonance (NMR) spectra were obtained in CD3Cl and CD3OD on Bruker Avance DRX 600 spectrometers at 600 MHz for hydrogen NMR (1H NMR) and 150 MHz for carbon NMR (13C NMR). Mass spectral data were performed on ion trap liquid chromatography/mass spectrum agilent. For column chromatography, silica gel (Merck, 70–230 mesh ASTM) and sephadex LH-20 (pharmacia) were used. Precoated silica gel 60 F-254 plates (Merck) were used for thin-layer chromatography (TLC). Reversed phase-18 (40–63 μm), 6 ml standard tubes (extraction tubes), Damstadt, Germany, were used for purification of the polar compounds. Spots were visualized by exposure to NH3 vapour, UV radiation and P-anisaldehyde/sulfuric acid.
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6

NMR Spectroscopy of Biological Samples

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All 1H NMR spectra were acquired at 300 K on a Bruker Avance DRX-600, operating at 600.13 MHz (Bruker Biospin, Wissembourg, Germany), equipped with an autosampler and an inverse 1H-13C-15N cryoprobe. For urine samples, 1H NMR spectra were acquired using a standard pulse sequence NOESY to suppress water resonance. A relaxation delay of 2 s and mixing time of 150 ms were used and 256 free induction decays (FIDs) were collected into 32 k data points using a spectral width of 20 ppm with an acquisition time of 1.36 s. 1H NMR spectra were acquired using the Carr-Purcell-Meiboom-Gill (CPMG) spin-echo pulse sequence with pre-saturation, with a total spin-echo delay (2 nt) of 240 ms to attenuate broad signals from proteins and lipoproteins. A total of 128 transients were collected into 32 k data points using a spectral width of 20 ppm, a relaxation delay of 2 s and an acquisition time of 1.36 s.
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7

PGRMC1 Binding Site Mapping

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For NMR measurements, 500 μM of GL-bound or heme-dimerized PGRMC1 were prepared in 50 mM phosphate buffer (pH 7.0) containing 5% D2O. NMR spectra were measured at 25 °C using a Bruker Avance DRX 600 spectrometer equipped with a triple resonance (1H/13C/15N) cryogenic probe. Transverse relaxation-optimized spectroscopy (TROSY) types of 2D 1H-15N heteronuclear single-quantum coherence (HSQC) spectra were measured. NMR data were processed using the program NMRPipe [36 (link)], and signal assignments were performed using the programs Kujira [37 (link)], and NMRView [38 (link)]. A docking model structure of the GL-bound PGRMC1 was calculated using the program HADDOCK 2.1 [39 (link)] using the crystal structure of PGRMC1 (PDB: 4X8Y) and the chemical shift data, and residues showing Δδ larger than 0.05 ppm were defined as the GL-binding site.
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8

NMR Spectroscopy in Biomolecular Analysis

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NMR spectra were recorded on a Bruker Avance DRX 600 equipped with a cryoprobe operating at 600 MHz for proton in D2O solution containing 1 mM sodium 3-trimethylsilyl [2,2,3,3-D4] propionate as a chemical shift reference for 1H spectra. Water was deionized and purified (Milli-Q, Millipore, Germany), whereas TRI Reagent® and all solvents were purchased from Sigma (Aldrich, Milan, Italy). Chromabond® HR-X resin was obtained from Macherey-Nagel GmbH (Düren, Germany). Ultrafiltration was carried out by Vivaspin 500 centrifugal concentrators (Sigma-Aldrich) with 3, 10 and 30 kDa molecular weight cut-off membrane of polyethersulfone (PES). Electrophoresis (SDS-PAGE) was performed by a mini gel apparatus BioRAD (Milan, Italy). To estimate the molecular mass, Precision Plus Protein™ Dual Color Standards (BioRad, Hercules, CA, USA) with 10 recombinant proteins of precise molecular weights (10–250 kD) were used.
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9

NMR and Mass Spectrometry Analysis

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Optical rotations were measured on a Jasco P2000 digital polarimeter (Jasco, Cremella, Italy). UV spectra were acquired on a Jasco V-650 spectrophotometer, NMR spectra were recorded on a Bruker Avance DRX 600 (Bruker, Milan, Italy) equipped with a cryoprobe operating at 600 MHz for protons. Chemical shift values are reported in ppm (δ) and referenced to internal signals of residual protons (CD3OD 1H δ3.34, 13C 49.0 ppm). High-resolution mass spectra were acquired on a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Milan, Italy); HPLC analyses have been performed on a Jasco system (PU-2089 Plus-quaternary gradient pump equipped with a Jasco MD-2018 Plus photodiode array detector). [1-13C]-acetate, [2-13C]-acetate and [1,2-13C2]-acetate, [1-13C]-glycolate (all sodium salts), and salicylhydroxamic acid (SHAM) were obtained from Sigma Aldrich (Milan, Italy). Solvents were purchased from VWR (Milan, Italy) and were HPLC-grade.
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10

Analytical and Preparative HPLC-MS Characterization

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Analytical HPLC was performed on a Shimadzu LC-10Avp series HPLC system (Shimadzu Deutschland GmbH, Germany) consisting of an autosampler, high pressure pumps, column oven and a diode array detector. HPLC conditions: C18 column (Eurospher 100-5 250 × 4.6 mm; Alltech, Germany) and gradient elution (acetonitrile (MeCN)/0.1% (v/v) trifluoroacetic acid (TFA) (H2O) 0.5/99.5 in 30 min to MeCN/0.1% (v/v) TFA 100/0, MeCN 100% (v/v) for 10 min), flow rate 1 ml min−1. All solvents used were of analytical or HPLC grade. liquid chromatography-mass spectrometry (LCMS) measurements were performed using an Exactive Orbit-rap High Performance Benchtop LC-MS Mass Spectrometer with an electrospray ion source (Thermo Fisher Scientific, Germany). HPLC conditions: C18 column (Betasil C18 3 μm 150 × 2.1 mm; Thermo Fisher Scientific, Germany) and gradient elution (MeCN/0.1% (v/v) HCOOH (H2O) 5/95 for 1 min, going up to 98/2 in 15 min, then 98/2 for another 3 min; flow rate 0.2 ml min−1; injection volume: 3 μl). Preparative HPLC was performed on a Shimadzu LC-8a series HPLC system with DAD. NMR spectra were recorded on a Bruker Avance DRX 600 instrument (Bruker BioSpin GmbH, Germany). Spectra were normalized to the residual solvent signals.
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