5 mm bbfo probe

The ¹⁹F NMR measurements were conducted at 28°C on a Bruker 400-MHz NMR spectrometer, equipped with a 5-mm BBFO probe. The experimental parameters were as follows: pulse angle, 90° (18.32 μsec); repetition rate, 1 sec; 172 K data set; 2,000 scans. All ¹⁹F data were processed using TopSpin and analyzed with MestReNova.

The ¹H NMR spectra were measured on a Bruker 700 MHz spectrometer using a 5 mm cryogenically cooled probe. The ³¹P spectra were acquired on a Bruker 500 MHz spectrometer using a 5 mm BBFO probe tuned to 202.46 MHz. The polar lipid extracts were prepared in a blend of CDCl₃/MeOH/CsEDTA(aq) according to Monakhova et. al. (2018) [39 (link)]. Semi-preparative HPLC was carried out on an Agilent 1200 Series HPLC (Santa Clara, CA, USA) equipped with a diode array detector. High resolution mass spectra were acquired using a Thermo Fisher Scientific (Waltham, MA, USA) Q Exactive^TM Hybrid Quadrupole-Orbitrap^TM Mass Spectrometer. GC-FID analysis was carried out on an Agilent Technologies 7890A GC spectrometer (Santa Clara, CA, USA).

Nanodisc samples were concentrated to ~200 μM in 280 μL in a Vivaspin-6 concentrator with a 30 kDa MWCO (Sartorius). 20 μL D₂O was added and gently mixed into the sample. ¹⁹F-NMR and ³¹P-NMR experiments were measured on a Bruker Avance III HD spectrometer operating at 600 MHz ¹H nutation frequency using Topspin 3.6.2 and equipped with a Bruker 5-mm BBFO probe. To make direct comparisons with previously published ¹⁹F-NMR data⁵⁰ , ¹⁹F-NMR spectra were measured at 280 K. ³¹P-NMR experiments were measured at 300 K to obtain improved spectral resolution. Temperatures were calibrated from a standard sample of 4% methanol in D₄-MeOH.
1-dimensional ¹⁹F data were recorded with a data size of 32k complex points, an acquisition period of 360 ms, 16k scans, 120 μs dwell time, and 0.3 s recycle delay for a total experimental time of about 3 hours per experiment. All ³¹P NMR experiments were acquired with an acquisition time of 900 ms, 2k scans, and 0.3 s recycle delay for a total experiment time of 42 min per experiment.
2-dimensional [¹⁹F,¹⁹F]-EXSY experiments were recorded with a data size of 120 and 8192 complex points in the indirect and direct dimensions, respectively. We recorded 256 scans for each experiment with 100 ms of mixing time.

Nanodisc samples were concentrated to ~200 μM in 280 μL in a Vivaspin-6 concentrator with a 30 kDa MWCO (Sartorius, Catalog Number: VS0621). 20 μL D₂O was added and gently mixed into the sample. ¹⁹F-NMR and ³¹P-NMR experiments were measured on a Bruker Avance III HD spectrometer operating at 600 MHz ¹H nutation frequency using Topspin 3.6.2 and equipped with a Bruker 5-mm BBFO probe. To make direct comparisons with previously published ¹⁹F-NMR data^{50 (link)}, ¹⁹F-NMR spectra were measured at 280 K. ³¹P-NMR experiments were measured at 300 K to obtain improved spectral resolution. Temperatures were calibrated from a standard sample of 4% methanol in D₄-MeOH.
1-dimensional ¹⁹F data were recorded with a data size of 32k complex points, an acquisition period of 360 ms, 16k scans, 120 µs dwell time, and 0.3 s recycle delay for a total experimental time of about 3 h per experiment. All ³¹P NMR experiments were acquired with an acquisition time of 900 ms, 2k scans, and 0.3 s recycle delay for a total experiment time of 42 min per experiment.
2-dimensional [¹⁹F,¹⁹F]-EXSY experiments were recorded with a data size of 120 and 8192 complex points in the indirect and direct dimensions, respectively. We recorded 256 scans for each experiment with 100 ms of mixing time.

The fecal and colorectal tissue samples were analyzed by ¹H NMR spectroscopy analysis. The samples were transferred into 5 mm NMR tubes individually on a Bruker AVANCE III spectrometer equipped at 600 MHz with a 5 mm-BBFO probe. ¹H NMR spectra were obtained by one dimensional NOESYPR1D pulse sequence. Free induction decays (FIDs) adopted a spectral width of 20 ppm with a mixing time of 100 ms and pulse delay time of 1.7 s, and they were collected with 128 transients into 32 k data points. An exponential function with a line-broadening factor of 0.3 Hz multiplied to all FIDs before Fourier transformation. The characteristic peaks of metabolites were detected according to the network database of metabolomics, including the Human Metabolome Database (HMDB¹) and Biological Magnetic Resonance Bank (BMRB²).

The method of ¹H-NMR analysis and data processing were described in a previous study [37 (link)]. Briefly, all ¹H-NMR spectra were recorded by Bruker 600 MHz AVANCE III spectrometer equipped with a 5 mm-BBFO probe at 25 °C. Shimming and proton pulse calibration was performed automatically for each sample before data acquisition. ¹H-NMR spectra were received using NOESYPR 1D pulse sequence with water suppression. Bruker Topspin 3.2 was used to process the data.
Free induction decays (FIDs) from ¹H-NMR of the fecal samples were multiplied by a 0.3 Hz exponential line broadening prior to Fourier Transformation. All NMR spectra were manually phased, baseline corrected and referenced to TSP (δ = 0.0) within MestReNova 12 (Mestrelab Research SL, Spain). The integral region of the spectrum was set between 0.0 and 9.0 ppm, with a spectral region of 4.5–5.0 ppm to eliminate the effects of imperfect water suppression. Due to the deviation of metabolite concentration in the fecal samples of each mouse, each bucket was internally normalized to the total sum of the spectral integrals prior to pattern recognition analysis. The characteristic peaks of all fecal metabolites were determined based on related literature [38 (link), 39 (link)] and the Biological Magnetic Resonance Bank (http://www.bmrb.wisc.edu/metabolomics) and Human Metabolome Database (http://www.hmdb.ca/).

NMR experiments were performed on a Bruker Avance III spectrometer operating at 9.4 Tesla (400 Mhz and 100.6 MHz for ¹H and ¹³C, respectively) using a Bruker 5 mm BBFO probe. Pulse widths were 14.1 and 10.5 μs for ¹H and ¹³C, respectively. Samples were dissolved in 400 μL of 50% pyridine-d₅ and 50% D₂O (pyridine-d₅ was used as reference, with highest field signals at 7.19 ppm for ¹H and 123.5 ppm for ¹³C), and all experiments were performed at 313 K. Standard experiments were performed using the Bruker software package: ¹H, ¹³C decoupled from proton, ¹³C JMOD, ¹H–¹H DQF-COSY, ¹H–¹³C HSQC, ¹H–¹³C HMBC, and ¹H–¹³C HSQC-TOCSY.
Acquisition parameters: ¹H spectral width 4400 Hz, ¹³C spectral width 24,000 Hz, repetition time 2 s. Double Quantum Filtered COSY experiment: 4K complex points × 512 increments, 16 scans per increment. HSQC experiment: 2K × 512 data set, 128 scans per increment. HMBC experiment: a typical value of 50 ms was used for the evolution of long-range coupling and a value of 3.4 ms for the low-pass J filter; 2K × 512 data set, 128 scans per increment. HSQC-TOCSY experiment: DIPSI2 scheme for homonuclear Hartman–Hahn mixing (80 ms), 2K × 512 data set, 128 scans per increment.