Aqueous metabolites were extracted from mouse gastrocnemius for NMR-based metabolomic analysis according to the protocol described previously [18 (link)–20 (link)]. All NMR experiments were performed at 25 °C on a Bruker Avance III 850 MHz spectrometer (Bruker BioSpin, Germany) equipped with a TCI cryoprobe. Both the unsupervised principal component analysis (PCA) and supervised partial least-squares discriminant analysis (PLS-DA) were applied to compare metabolic profiles of gastrocnemius among the four groups of the NOR, CAC, AM, and KD mice by using the SIMCA 14.1 software (MKS Umetrics AB, Sweden). The metabolic pathway analysis was performed to identify significantly altered metabolic pathways (significant pathways) on the MetaboAnalyst 4.0 webserver (https://www.metaboanalyst.ca ). This webserver was also used to obtain heat-map plots of relative metabolite levels, and the Pearson’s correlation coefficients between catabolic protein expressions and metabolite levels.
Avance 3 850 mhz spectrometer
Manufactured by Bruker
Sourced in Germany
The Avance III 850 MHz spectrometer is a high-performance nuclear magnetic resonance (NMR) instrument produced by Bruker. It is designed to provide high-resolution, sensitive, and accurate measurements of molecular structures and dynamics.
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5 protocols using avance 3 850 mhz spectrometer
1
Metabolomic Analysis of Mouse Gastrocnemius
2
NMR Structural Characterization of Peptides
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The hd1 and hd2 peptides were dissolved in H2O/D2O (90%/10%) and H2O/CD3CN (50%/50%) with a final concentration of 1.3 mM and 0.8 mM, respectively. All NMR experiments were conducted at 298 K on a Bruker AVANCE III 850 MHz spectrometer equipped with a 5 mm z-gradient 1H/13C/15N TCI cryogenic probe. Two-dimensional (2D) 1H–1H TOCSY (80 ms mixing time) and 1H–13C/15N HSQC spectra were measured for resonance assignments. 2D 1H–1H NOESY experiments with a mixing time of 200 or 300 ms were performed to extract 1H–1H distance. The phi and psi backbone torsion angles were predicted by TALOS+ using chemical shifts of HN, HA, CA, CB, and N39 (link). The Xplor-NIH (version 2.53) program was used for the structure determination and refinement40 . The intra- and inter-molecular hydrogen bond distance constraints inferred from the preliminarily calculated structures were applied in the late stage of structure determination. All NMR spectra were processed using TopSpin 3.5 and analyzed using NMRFAM-SPARKY41 (link). 1H chemical shifts were referenced to DSS, and 13C/15N chemical shifts were referenced indirectly to DSS.
3
NMR Characterization of Protein Samples
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The
15N- and
13C/
15N-labeled NMR samples were prepared with 0.8 mM
MtPC in the NMR buffer (200 mM NaCl, 50 mM Na
2HPO
4-NaH
2PO
4, pH 6.2, 0.02% NaN
3, and 10% D
2O). All NMR experiments were performed at 298 K on a Bruker Avance III 850 MHz spectrometer (Bruker, Rheinstetten, Germany) equipped with a TCI triple-resonance cryoprobe. The two- and three-dimensional spectra of
1H-
15N HSQC,
1H-
13C HSQC, HNCACB, CBCA(CO)NH, HNCA, HN(CO)CA, HNCO, and HN(CA)CO were recorded for backbone resonance assignments. For
15N T
1 measurements, the delay times were set to 10, 50, 100 (×2), 200, 400, 600, 800 (×2), 1200, 1600, and 2000 ms. For
15N T
2 measurements, the delay times were set to 17.0, 33.9 (×2), 50.9, 67.8, 84.8, 102, 119, 136 (×2), 153, and 170 ms. The repeated experiments were used to estimate uncertainties of T
1 and T
2 values. For {
1H}-
15N NOE experiments, a delay of 2 s was followed by
1H saturation of 3 s, and in the control experiments without
1H saturation, a total delay of 5 s was applied. All NMR spectra were processed using NMRPipe and analyzed using NMRFAM-SPARKY.
15N- and
13C/
15N-labeled NMR samples were prepared with 0.8 mM
MtPC in the NMR buffer (200 mM NaCl, 50 mM Na
2HPO
4-NaH
2PO
4, pH 6.2, 0.02% NaN
3, and 10% D
2O). All NMR experiments were performed at 298 K on a Bruker Avance III 850 MHz spectrometer (Bruker, Rheinstetten, Germany) equipped with a TCI triple-resonance cryoprobe. The two- and three-dimensional spectra of
1H-
15N HSQC,
1H-
13C HSQC, HNCACB, CBCA(CO)NH, HNCA, HN(CO)CA, HNCO, and HN(CA)CO were recorded for backbone resonance assignments. For
15N T
1 measurements, the delay times were set to 10, 50, 100 (×2), 200, 400, 600, 800 (×2), 1200, 1600, and 2000 ms. For
15N T
2 measurements, the delay times were set to 17.0, 33.9 (×2), 50.9, 67.8, 84.8, 102, 119, 136 (×2), 153, and 170 ms. The repeated experiments were used to estimate uncertainties of T
1 and T
2 values. For {
1H}-
15N NOE experiments, a delay of 2 s was followed by
1H saturation of 3 s, and in the control experiments without
1H saturation, a total delay of 5 s was applied. All NMR spectra were processed using NMRPipe and analyzed using NMRFAM-SPARKY.
4
NMR Structural Determination of HuPrP
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To perform backbone and side chain resonances and to determine the solution structures of the HuPrP(G127V) and WT HuPrP proteins, we recorded a suite of 2D/3D heteronuclear NMR spectra at 25 °C on a Bruker Avance III 850-MHz spectrometer (magnetic field strength is 19.97 T) with a 1H/13C/15N triple-resonance cryogenic probe (TCI). These 3D NMR spectra included HNCACB, CBCA(CO)NH, HNCA, HNCOCA, HNCO, HN(CA)CO, HBHA(CO)NH, H(CCCO)NH, CC(CO)NH and (H)CCH-TOCSY. A mixing time of 120 ms was used for both 15N-edited NOSEY-HSQC and 13C-edited NOESY-HSQC experiments. All NMR spectra were processed by NMRPipe software82 (link) and analysed with CARA software83 .
5
NMR Spectroscopy for Protein Structural Assignments
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All NMR experiments were carried out at 298 K on a Varian Inova 600 MHz spectrometer equipped with a 5 mm HCN cryogenic probe and a Bruker Avance III 850 MHz spectrometer equipped with a 5 mm HCN conventional room temperature probe. The collected NMR data included: 2D 1H-15N HSQC, 1H-13C HSQC, 3D HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, HBHA(CO)NH, H(CCO)NH, and C(CO)NH for backbone assignments, and 2D 1H-13C HSQC (aliphatic and aromatic), 3D H(C)CH-TOCSY, H(C)CH-COSY, (H)CCH-TOCSY, and 4D CC NOESY for side chain assignments. The stereospecific assignments of isopropyl methyl groups of Val and Leu residues were determined from 2D constant-time 1H-13C HSQC spectrum for the NC5 sample [33 (link)]. The data were processed using the NMRPipe [34 (link)] and analyzed with the Sparky program [35 (link)]. The backbone and side-chain resonances were automatically assigned using the PINE server from NMRFAM [36 (link)], and subsequently validated and corrected manually. All assignments were further confirmed from NOESY spectra, including 3D 15N-edited NOESY-HSQC and 13C-edited NOESY-HSQC. Chemical shift assignments have been deposited in the BioMagResBank (BMRB accession number 16688).
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