A 500 µL aliquot of each (thawed) hemolymph or plasma fraction was prepared for the NMR analysis by adding 150 µL of a 0.8-mM trimethylsilylpropanoic acid (TSP) solution in D2O. The mussel tissues mentioned above were extracted according to the Folch method (Folch et al., 1957 (link)) using a 2:1 chloroform–methanol (v/v) solvent mixture at a solvent–tissue ratio of 8 mL/g. After addition of 4 mL deionized H2O and centrifugation at 5,400 × g for 15 min at 4°C, the methanol/water mixture was separated from the chloroform fraction. Both phases were evaporated and immediately stored at −20°C. All polar extracts were resuspended in 650 µL of D2O containing 0.8 mM TSP, transferred into a 5-mm NMR tube, and immediately analyzed. NMR spectra were acquired with a Bruker Avance Neo 600 MHz spectrometer (Bruker BioSpin, Karlsruhe, Germany) equipped with a Prodigy cryoprobe and using Topspin 4.0 software, applying a noesypr1D pulse sequence with spectral width 11 kHz, acquisition time 2.75 s, 64 scans, relaxation delay 4 s, and four dummy scans. All spectra were processed with an ACD NMR processor 12.1 (shortened to ACD, ACD Labs).
Avance neo 600 mhz spectrometer
Manufactured by Bruker
Sourced in Germany, Switzerland
The Avance NEO 600 MHz spectrometer is a high-performance nuclear magnetic resonance (NMR) instrument designed for analytical applications. It operates at a frequency of 600 MHz and offers advanced capabilities for the characterization and analysis of chemical samples.
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Lab products found in correlation
Topspin 4, by Bruker (2 mentions)
Prodigy cryoprobe, by Bruker (1 mentions)
Topspin 3, by Bruker (1 mentions)
Avance spectrometer, by Bruker (1 mentions)
Sodium chloride (nacl), by Carl Roth (1 mentions)
Hepes, by Carl Roth (1 mentions)
Inova 600 mhz, by Bruker (1 mentions)
Iraffinity 1s fourier transform infrared spectrometer, by Shimadzu (1 mentions)
Finnigan ltq orbitrap elite, by Thermo Fisher Scientific (1 mentions)
Mcp 500, by Anton Paar (1 mentions)
Sephadex lh 20, by GE Healthcare (1 mentions)
Chirascan spectropolarimeter, by Applied Photophysics (1 mentions)
Silica gel gf254 plate, by Qingdao Marine Chemical (1 mentions)
Stddiffesgp 3, by Bruker (1 mentions)
13 protocols using avance neo 600 mhz spectrometer
1
Metabolomic Analysis of Mussel Hemolymph and Tissues
2
Structure Elucidation of Fucoxanthin by NMR
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The structure of fucoxanthin was determined by nuclear magnetic resonance (NMR) spectra recorded on a Bruker Avance Neo 600 MHz spectrometer (Bruker, Coventry, UK), with CDCl3 as the solvent and TMS as the internal standard. 1H NMR spectra were measured at 600 MHz, and 13C was measured at 150 MHz at the Institute of Chemistry, VAST. The structure of fucoxanthin was determined by comparing the NMR data with the published standard [40 (link)].
3
NMR-based Urine Sample Preparation
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All collected samples were centrifuged (1.5 mL) at 10,000× g for 30 min to remove fine particles. Then, 600 µL of supernatant was mixed with 300 µL 1H-NMR buffer as described before [77 (link)] and stored at −25 °C until analysis. Before analysis, samples were thawed at room temperature and transferred into a 5 mm NMR tube. TSP was used as an internal standard for quantification, as described before [77 (link)].
A Bruker Avance NEO 600 MHz spectrometer equipped with a 24-sample SampleCase autosampler and a 5 mm HCN Cold probe was used for the acquisition of NMR spectra at 25 °C. The 1H NMR spectra of the samples were recorded with a spectral width of 9.0 kHz, relaxation delay of 2.0 s, 32 scans and 32 K data points. A double-pulsed field gradient spin echo (DPFGSE) pulse sequence was used for water suppression. Total correlated spectrum (TOCSY) was measured with 1H spectral widths of 7.0 kHz, 4096 complex points, a relaxation delay of 1.5 s, 32 transients and 144 time increments. An exponential and cosine-squared function were used for apodization. Zeros were filled before Fourier transform. TopSpin v. 4.0.9 software (Bruker, Billerica, MA, USA) was used for processing urine NMR spectra [4 (link),5 (link),76 (link),78 (link)]. AlpsNMR R package was used for the visualization of example spectra [79 (link)].
A Bruker Avance NEO 600 MHz spectrometer equipped with a 24-sample SampleCase autosampler and a 5 mm HCN Cold probe was used for the acquisition of NMR spectra at 25 °C. The 1H NMR spectra of the samples were recorded with a spectral width of 9.0 kHz, relaxation delay of 2.0 s, 32 scans and 32 K data points. A double-pulsed field gradient spin echo (DPFGSE) pulse sequence was used for water suppression. Total correlated spectrum (TOCSY) was measured with 1H spectral widths of 7.0 kHz, 4096 complex points, a relaxation delay of 1.5 s, 32 transients and 144 time increments. An exponential and cosine-squared function were used for apodization. Zeros were filled before Fourier transform. TopSpin v. 4.0.9 software (Bruker, Billerica, MA, USA) was used for processing urine NMR spectra [4 (link),5 (link),76 (link),78 (link)]. AlpsNMR R package was used for the visualization of example spectra [79 (link)].
4
NMR Analysis of Peptides
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For NMR analysis, lyophilized peptides were resuspended at a concentration of 2 mM (PnID A) or 1 mM (PnID B) in 95% H2O/5% D2O with 0.2 mM 2,2-dimethylsilapentane-5-sulfonic acid (DSS) as a chemical shift reference. The samples were adjusted with HCl to pH 4.0.
All NMR spectra were collected with a sample temperature of 5 °C using a Bruker (Billerica, MA) Avance NEO 600 MHz spectrometer with a room-temperature TXI probe. Proton chemical shift assignments were made using 1H-1H TOCSY (60 ms mixing time) and 1H-1H NOESY spectra (100 ms mixing time) experiments. Carbon and nitrogen resonances were assigned using the natural abundance signals measured by 1H-13C HSQC and 1H-15N BEST-HSQC experiments. NOE-based distance constraints were generated from 1H-1H NOESY spectra (600 ms mixing time). 3JHN-Hα values were measured directly from 1D 1H spectra. NMR data were processed using TopSpin 4.1.4 (Bruker) and assigned using CCPN Analysis 2.5.2 [26 (link)].
All NMR spectra were collected with a sample temperature of 5 °C using a Bruker (Billerica, MA) Avance NEO 600 MHz spectrometer with a room-temperature TXI probe. Proton chemical shift assignments were made using 1H-1H TOCSY (60 ms mixing time) and 1H-1H NOESY spectra (100 ms mixing time) experiments. Carbon and nitrogen resonances were assigned using the natural abundance signals measured by 1H-13C HSQC and 1H-15N BEST-HSQC experiments. NOE-based distance constraints were generated from 1H-1H NOESY spectra (600 ms mixing time). 3JHN-Hα values were measured directly from 1D 1H spectra. NMR data were processed using TopSpin 4.1.4 (Bruker) and assigned using CCPN Analysis 2.5.2 [26 (link)].
5
NMR Analysis of TACC3 Peptides
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Side chain assignments of TACC3 519–563 & 519–540 were obtained from 15N/13C‐labelled samples expressed as described before (Burgess et al, 2015 ) in 20 mM KPO4 pH 7.0, 50 mM NaCl, 1 mM DTT, 0.02% (w/v) sodium azide at a protein concentration of 300 μM. 3D H(CCCO)NH, (H)C(CCO)NH, 13C HCCH‐TOCSY, 13C NOESY‐HSQC and 13C HSQC spectra were recorded for these peptides alone and in complex with Aurora‐A 122–403 D274N on a Bruker Avance spectrometer operating at a 1H frequency of 700 MHz and a temperature of 298K. Spectra were recorded and processed using Bruker TopSpin™ 3.2 software (Bruker Biospin AG: Fällanden, Switzerland) and analysed using CCPN analysis.
For analysis of TACC3 549–570 helicity, synthetic peptides were weighed in to give a sample concentration of 2 mM in 20 mM Tris pH 7.2 and 50 mM NaCl. Spectra were recorded at a temperature of 290 K on a Bruker Avance‐Neo 600 MHz spectrometer equipped with a prodigy cryoprobe. Peptides were assigned using 2D NOESY, TOCSY and 13C HSQC spectra recorded with watergate and gradient coherence selection, respectively. Assignment and chemical shift analysis was performed in CCPN analysis version 2.4. This software does not have standard random coil chemical shifts values for phospho‐serine so it was not possible to calculate secondary chemical shift values for pS558 in the phosphorylated peptide.
For analysis of TACC3 549–570 helicity, synthetic peptides were weighed in to give a sample concentration of 2 mM in 20 mM Tris pH 7.2 and 50 mM NaCl. Spectra were recorded at a temperature of 290 K on a Bruker Avance‐Neo 600 MHz spectrometer equipped with a prodigy cryoprobe. Peptides were assigned using 2D NOESY, TOCSY and 13C HSQC spectra recorded with watergate and gradient coherence selection, respectively. Assignment and chemical shift analysis was performed in CCPN analysis version 2.4. This software does not have standard random coil chemical shifts values for phospho‐serine so it was not possible to calculate secondary chemical shift values for pS558 in the phosphorylated peptide.
6
Probing NGF-ATP Interactions by NMR
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The 1H STD and the tr‐NOESY experiments were recorded on a Bruker Avance Neo 600 MHz spectrometer using a cryoprobe at 30°C on samples containing 10 μM unlabeled rh‐proNGF in 50 mM Tris‐d11 and 50 mM NaCl in D2O, pD 7.3 buffer. The spectra were recorded at an ATP/rh‐proNGF ratio of 100:1, that is a protein concentration of 10 μM (with respect to the dimer) and 1 mM ATP. To investigate the effect of the cation, MgCl2 was added to the samples, at the following concentrations: 0, 10, 100 μM, and 1, 2 mM.
The 1H STD ligand epitope mapping experiments (Mayer & Meyer, 2001 (link)) were performed under quantitative conditions, considering the non‐uniform relaxation properties of ATP. Errors in the STD amplification factor were estimated according to the formula: STD amplification factor absolute error = STD amplification factor × ((NSTD/ISTD)2 + (NREF/IREF)2)1/2 (McCullough et al., 2012 (link)). NSTD and NREF are noise levels in STD and reference spectra. ISTD and IREF are signal intensities in STD and reference spectra.
The tr‐NOESY spectra were recorded with a 6578 Hz sweep width, 4096 data points in t2, 48 scans, 128 complex data points in t1, a mixing time of 350 ms, and a relaxation delay of 1.5 s.
Please find experimental details in AppendixS1 .
The 1H STD ligand epitope mapping experiments (Mayer & Meyer, 2001 (link)) were performed under quantitative conditions, considering the non‐uniform relaxation properties of ATP. Errors in the STD amplification factor were estimated according to the formula: STD amplification factor absolute error = STD amplification factor × ((NSTD/ISTD)2 + (NREF/IREF)2)1/2 (McCullough et al., 2012 (link)). NSTD and NREF are noise levels in STD and reference spectra. ISTD and IREF are signal intensities in STD and reference spectra.
The tr‐NOESY spectra were recorded with a 6578 Hz sweep width, 4096 data points in t2, 48 scans, 128 complex data points in t1, a mixing time of 350 ms, and a relaxation delay of 1.5 s.
Please find experimental details in Appendix
7
Synthesis and Characterization of Novel Organic Compounds
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Solvents and reagents were used as received unless otherwise noted. Thin-layer chromatography (TLC) was performed on silica gel 60 F254 (Merck, KGaA, Darmstadt, Germany). The products of the reactions were purified by normal chromatography column using Silica Gel Kiesegel 60 (70–230 mesh). Yields corresponded to isolated compounds. Melting points were determined in Kofler melting apparatus and values are uncorrected. All synthesized compounds were characterized by 1H NMR and 13C NMR spectroscopy. NMR experiments were obtained at 25 °C on a Bruker Avance at 400 MHz spectrometer, a Bruker Avance NEO 600 MHz spectrometer or a Bruker DPX 200 MHz spectrometer (Bruker, Billerica, MA, USA). Chemical shifts (δ) are reported in parts per million (ppm) in CDCl3 solution, if not otherwise specified. The following abbreviations are used to indicate multiplicity: s—singlet; d—doublet; t—triplet; q—quartet; quin—quintet; m—multiplet. Exact mass analyses were obtained by mass spectrometer Ion-Mobility QTof Agilent 6560 coupled with UHPLC 1290 Infinity II Agilent (UHPLC Agilent Technologies, Santa Clara, CA, USA).
8
Synthesis and Characterization of Pyridine and Chromone Aldehydes
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Both pyridine-4-carboxaldehyde and chromone-3-carboxaldehyde were purchased from HEOWNS Biochem Technologies LLC, Tianjin, China. Unless otherwise noted, all other reagents and solvents were commercially available and used without further purification. NMR spectra were recorded using a Bruker Avance NEO 600 MHz spectrometer (at 600 MHz for 1H NMR or 150 MHz for 13C NMR; Rheinstetten, Germany). A WQF-510A FT-IR spectrometer (Beijing Rayleigh Analytical Instrument Co., Ltd., Beijing, China) recorded the infrared spectroscopy in KBr discs in the 400–4000 cm−1 region. The melting points were measured on a Beijing Cossim X-5T micro-melting point apparatus (Beijing Century Letter Scientific instrument Co., Ltd., Beijing, China). HRMS (high-resolution mass spectra) was performed using an Aglient 7250 spectrometer (Santa Clara, CA, USA). Absorption spectra and fluorescence spectra were obtained using a TU-1901 UV-Vis spectrophotometer (Beijing Puxi General Instrument Co., Ltd., Beijng, China) and a Varian Cary Elipses spectro fluorophotometer (Palo Alto, CA, USA), respectively.
9
Structural Analysis of p53 Binding
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Proteins were purified into 20 mM Hepes (Carl Roth, Karlsruhe, Germany) pH 7.0, 50 mM NaCl (Carl Roth, Karlsruhe, Germany), 2 mM TCEP, and 0.04% (w/v) NaN3. All binding experiments were performed at 25 °C on a Bruker Avance Neo 600 MHz spectrometer (Bruker, Rheinstetten, Germany) equipped with a triple-resonance probehead. 1H-15N HSQC spectra (hsqcetfpf3gpsi, 16 scans, 128 points in F1, 1024 points in F2) were recorded for the titrations of 50 µM 15N-p53 1−312, 15N-p53DBD, 15N-p53TAD1, 15N- p531−94, and 15N-p53TAD2 with one stoichiometric equivalent of either PR25 or GR25. Reference 1H-15N HSQC spectra were recorded for 15N-p53 1−312, 15N-p53DBD, 15N-p53TAD1, 15N- p531−94, and 15N-p53TAD2 without a binding partner. Data analysis was performed using Bruker Topspin 4.02 (Bruker, Rheinstetten, Germany) and ccpnmr (version 2.5.) [70 (link)]. CSPs of p531−94 and p53TAD2 1H-15N cross-peaks upon binding to PR25/GR25 were calculated using the following equation:
10
NMR Experiments with Ligand Screening
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NMR experiments were performed at 25 °C on a either a Varian 900 MHz DD2, Varian INOVA 600 MHz or Bruker Avance Neo 600 MHz spectrometer. Samples for NMR were dissolved in sodium phosphate (20 mM, pH 6.5) and 90% H2O/10% D2O, with DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) (80 μM) added as an internal reference 89 (link),90 (link). For ligand screening, protein concentration was 100 μM and ligands were added to a concentration of 200–500 μM. For chemical shift assignments, relaxation rate measurements and structure calculations, protein concentrations were in the 400–700 μM range. All NMR experiments were collected using non uniform sampling methods91 using the Poisson-gap sampling schemes implemented by Hyberts et al.92 (link) with a sampling density of 25–50%. Data were processed using the istHMS package v211193 (link),94 (link) in combination with NmrPipe95 (link) and analyzed using Ccpnmr Analysis v 2.4.296 (link).
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