Aviii 600 mhz spectrometer

Serum NMR experiments were performed on a Bruker AV III 600 MHz spectrometer (Bruker Bio spin, Corporation, Billerica, MA, USA) equipped with an inverse cryoprobe operating at 600.13 MHz as our described method [22 (link)]. All NMR spectra of samples were acquired using a standard Bruker noesygppr1d pulse sequence and a total of 64 scans were collected into 32,768 data points over a spectral width of 8000.00 Hz. The first increment of a 2D-¹H, ¹H-NOESY pulse sequence was utilized for the acquisition of ¹H-NMR data and for suppressing the solvent signal. Experiments used a 100 ms mixing time along with a 990 ms pre-saturation (~80 Hz gammaB1), and recycle delay is 1s. Spectra were collected at 25 °C, with a total of 128 scans over a period of 15 min. The data was analyzed using Chenomx software (Version 8.1, Alberta, AB, Canada) to conduct phasing and baseline correction with a consistent setting. The signal at δ 4.76–4.97 (water) in serum samples was excluded. Spectral data in the region of δ 0.50 to δ 10.00 were then normalized using the total spectral area normalization method and binned into a bin spectral width of 0.04 ppm. Spectral intensities were then scaled to sodium 3-(trimethylsilyl) propionate-2,2,3,3-d4 (TSP). Metabolic profiling and peak identification were performed by Anachro Technologies Inc. (Wuhan, China) [22 (link),36 (link),37 (link)].

The biotransformed products were isolated using a Waters prep-HPLC system (Waters, USA) with a Waters Sunfire Prep C₁₈ OBD^™ column (19 mm×50 mm, 5 μm). Isocratic elution was performed at room temperature using a mixture of H₂O/ACN (56:44, v/v) for product 1, H₂O/ACN (48:52, v/v) for product 2, H₂O/ACN (54:46, v/v) for product 3 and H₂O/ACN (54:46, v/v) for product 4, respectively. The flow rate was 15 mL/min, and the UV detection was at 203 nm. The injection volume was set at 200 µL. The purity of the products were evaluated by HPLC in an Agilent 1200 series HPLC system (Agilent Technologies, USA). The separation conditions were the same as that used in the HPLC analysis. The ¹³ C NMR spectra of the purified products were recorded on a Bruker AVIII 600 MHz spectrometer at an operating frequency of 151 MHz (Bruker BioSpin, Germany).

¹⁵N-edited HSQC spectra were recorded using the standard pulse sequence with WATERGATE solvent suppression^{64 (link),65 (link)}. Chemical shifts were assigned using standard HNCA, HN(CO)CA, HNCO, HN(CA)CO, CBCA(CO)NH, and HNCACB experiments acquired with non-uniform sampling (25%)^{66 (link),67 (link)}. All data were recorded using a Bruker AVIII 600 MHz spectrometer with a cryogenic probe.
The data were processed using NMRPipe^{68 (link)}. Chemical shifts were assigned using the automated assignment scheme^{69 (link)} implemented in NMRFAM-Sparky^{70 (link)}, and then confirmed manually. Secondary chemical shift analysis was performed using Kjaergaard et al.’s database of random-coil shifts^{71 (link)}. Secondary structure populations were estimated using δ2D^{34 (link)}. No chemical shifts were missing around the critical α-helical region, such that the secondary structure estimates for the constructs were made using the same number of chemical shifts.

All NMR experiments were performed on Bruker AV III 600 MHz spectrometer (Bruker Biospin, Milton, Canada) equipped with an inverse cryoprobe operating at 600.13 MHz. All NMR spectra of samples were acquired using a standard Bruker noesygppr1d pulse sequence, and a total of 256 scans were collected into 32768 data points over a spectral width of 8000.00 Hz.

STD NMR spectra were recorded using 3 mm tubes on a Bruker AVIII-600 MHz spectrometer equipped with a room temperature probe head at 288 K. Data was processed with TOPSPIN 3.0 (Bruker). The sample contained 2 mM GM1 oligosaccharide (Elicityl, France) or 20 μM TSPyV VP1 and 2 mM GM1 oligosaccharide, respectively, in 20 mM K₂HPO₄/ KH₂PO₄ pH 7.4, 150 mM NaCl, 99%D₂O. Off- and on-resonance frequencies were -30 ppm and 7.3 ppm, respectively. The irradiation power and length of the selective pulse train was 57 Hz and 2 s, respectively. In order to suppress residual protein resonances a continuous-wave spin-lock pulse with a strength of 3.2 kHz was employed. The relaxation delay was 3 s and a total of 5 k scans were recorded. Spectra were multiplied with a Gaussian window function prior to Fourier transform and referenced to the a-D-Glc anomeric proton as an internal standard [68 (link)].