Table 2.
Type of samples, pre‐analytical procedures, NMR sample preparation, NMR spectral acquisition.
| Type of | Pre‐analytical procedures[a] | Analytical procedures | |
|---|---|---|---|
| sample | NMR sample preparation[b] | NMR spectral acquisition[c] | |
| URINE | Collect the first urine of the morning after a minimum of 8 h fasting. Specify if collected in different daytimes or not fasting. Centrifuge the urine within 120 min after the collection at 1000–3000 RCF for 5 min at +4 °C and/or filtrate the samples by 0.20 μm cut‐off filter. Recover the urine in sterile condition making 1 mL aliquots. Store at −80 °C.29 |
Thaw at room temperature and shake. Centrifuge at 14000 RCF for 5 min at 4 °C.30 Add 630 μl of sample to 70 μL of potassium phosphate buffer (1.5 m K2HPO4, 100 % (v/v) 2H2O, 2 mm NaN3, 5.8 mm TMSP; pH 7.4).[d] Transfer 600 μL of each mixture into a 5 mm NMR tube.30 |
31Recommended magnetic field: 600 MHz Recommended acquisition temperature: 300 K 1D NOESY‐presat (Bruker: noesygppr1d) Scans: 32 Data points: 65 536 Spectral width: 12 019.230 Hz Acquisition time: 2.73 s Relaxation delay: 4 s Mixing time: 0.01 s 2D J‐RES (Bruker: jresgpprqf) Scans: 2 Data points direct dimension (F2): 8192 Data points indirect dimension (F1): 40 Spectral width direct dimension (F2): 10 026.738 Hz Spectral width indirect dimension (F2): 78.000 Hz Increment for delay: 12820.51 μs Acquisition time direct dimension (F2): 0.41 s Acquisition time indirect dimension (F1): 0.26 s Relaxation delay: 2 s |
| BLOOD |
SERUM
Collect blood into anticoagulant free tubes. Allow the blood to clot in an upright position for 30–60 min at RT. Spin centrifuge within 30 min from collection at 1500 RCF for 10 min at RT.29 PLASMA Collect blood into tubes contain an anticoagulant (preferred EDTA; avoid heparin). Centrifuge within 30 min from collection at 820 RCF for 10 min at 4 °C.29 |
Thaw at room temperature and shake. Add 350 μl of sample to 350 μL of sodium phosphate buffer (70 mm Na2HPO4; 20 % (v/v) 2H2O, 6.1 mm NaN3; 4.6 mm TMSP; pH 7.4).[d] Transfer 600 μL of each mixture into a 5 mm NMR tube.30 |
31Recommended magnetic field: 600 MHz Recommended acquisition temperature: 310 K 1D NOESY‐presat (Bruker: noesygppr1d) Scans: 32 Data points: 98 304 Spectral width: 18 028.846 Hz Acquisition time: 2.73 s Relaxation delay: 4 s Mixing time: 0.01 s 1D CPMG (Bruker: cpmgpr1d) Scans: 32 Data points: 73 728 Spectral width: 12 019.230 Hz Acquisition time: 3.07 s Relaxation delay: 4 s Total spin‐echo delay: 80 ms 1D Diffusion‐edited (Bruker: ledbpgppr2s1d) Scans: 32 Data points: 98 304 Spectral width: 18 028.846 Hz Acquisition time: 2.73 s Relaxation delay: 4 s Square gradient strength: 80 % of the maximum gradient strength (53.5 G cm−1) Square gradient length: 1.5 ms Diffusion time: 0.119 s 2D J‐RES (Bruker: jresgpprqf) For all the parameters see urine. |
| For both serum and plasma: recover supernatant in sterile condition making 0.5 mL aliquots. Store at −80 °C.29 | |||
| SALIVA | Collect saliva after refraining from eating, drinking, smoking, or using oral hygiene products for at least 1 h. Rinse the mouth twice with water before spitting the saliva in sterile tubes making 1 mL aliquots. Freeze asap at −20 °C and then store in liquid nitrogen.32 |
Thaw at room temperature and shake. Centrifuge at 14000 RCF for 5 min at +4 °C. Add 630 μl of sample to 70 μL of potassium phosphate buffer (1.5 m K2HPO4, 100 % (v/v) 2H2O, 2 mm NaN3, 5.8 mm TMSP; pH 7.4). Transfer 600 μL of each mixture into a 5 mm NMR tube.33 |
Recommended magnetic field: 600 MHz Recommended acquisition temperature: 300 K 1D NOESY‐presat (Bruker: noesygppr1d) Scans: 128 For all the other parameters see urine. 2D J‐RES (Bruker: jresgpprqf) For all the parameters see urine. |
| CSF | Collect CSF via lumbar puncture in sterile polypropylene tubes. Centrifuge asap at 2000 RFC for 10 min at RT. Collect the supernatant in sterile condition making 0.5 mL aliquots. Store at −80 °C.34 |
Thaw at room temperature and shake. Add 750 μL of sample to 150 μL of potassium phosphate buffer (0.9 m K2HPO4, 60 % (v/v) 2H2O, 1.2 mm NaN3, 3.5 mm TMSP; pH 7.4).[d] Transfer 600 μL of each mixture into a 5 mm NMR tube. |
Recommended magnetic field: 600 MHz Recommended acquisition temperature: 300 K 1D NOESY‐presat (Bruker: noesygppr1d) Scans: 256 For all the other parameters see blood. 2D J‐RES (Bruker: jresgpprqf) For all the parameters see urine. |
| EBC | Collect EBC after refraining from eating for at least 3 h using a condenser equipped with a saliva trap. Rinse the mouth twice with water before breathing through a mouthpiece for 15 min making a 1.5 mL aliquot. Transfer the EBC into glass vials closed with 20 mm butyl rubber lined with polytetrafluoroethylene septa and crimped with perforated aluminum seals. Before sealing, remove volatile substances by a gentle nitrogen gas flow for 3 min. Freeze asap in liquid nitrogen and then store at −80 °C.35 |
Thaw at room temperature and shake. Centrifuge at 14000 RCF for 5 min at +4 °C. Add 540 μl of sample to 60 μL of 2H2O containing 5.8 mm TMSP. Transfer 600 μL of each mixture into a 5 mm NMR tube. |
Recommended magnetic field: 600 MHz Recommended acquisition temperature: 300 K 1D NOESY‐presat (Bruker: noesygppr1d) Scans: 512 For all the other parameters see urine. 2D J‐RES (Bruker: jresgpprqf) For all the parameters see urine. |
| FECES | Collect feces in sterile containers. Add 2 mL of PBS/2H2O buffer (0.1 m, pH 7.4) to 1 g of each feces sample and homogenize the mixture by vortexing for 60 s.36 Sonicate for 30 min and centrifuge at 10000 RCF for 5 min at RT. Collect the supernatant in sterile condition making 1 mL aliquots. Store at −80 °C storage.37 |
Thaw at room temperature and shake. Centrifuge at 14000 RCF for 5 min at +4 °C. Add 630 μl of sample to 70 μL of potassium phosphate buffer (1.5 m K2HPO4, 100 % (v/v) 2H2O, 2 mm NaN3, 5.8 mm TMSP; pH 7.4). Transfer 600 μL of each mixture into a 5 mm NMR tube. |
Recommended magnetic field: 600 MHz Recommended acquisition temperature: 300 K 1D NOESY‐presat (Bruker: noesygppr1 d) Scans: 128–256 For all the other parameters see urine. 2D J‐RES (Bruker: jresgpprqf) For all the parameters see urine. |
| CELLS |
ENDO‐METABOLOME
Place cell plates into ice and rinse with PBS. Scrape and collect cells with PBS supplemented with 1 % Protease Inhibitor Cocktail and 1 % Phosphatase Inhibitor Cocktail. Sonicate and ultra‐centrifugate for 1 h at 4 °C. Collect the supernatant in sterile condition making 0.6 mL aliquots. Store at −80 °C freezer.23 |
Thaw in ice and shake. Add 540 μl of sample to 60 μL of 2H2O containing 5.8 mm TMSP. Transfer 600 μL of each mixture into a 5 mm NMR tube.23 |
Recommended magnetic field: 900 MHz Recommended acquisition temperature: 300 K 1D NOESY‐presat (Bruker: noesygppr1d) Scans: 128–256 Data points: 110 060 Spectral width: 17 942.584 Hz Acquisition time: 3.07 s Relaxation delay: 4 s Mixing time: 0.01 s |
|
1D CPMG
(Bruker: cpmgpr1d) Scans: 128–256 Data points: 110 060 Spectral width: 17 942.584 Hz Acquisition time: 3.07 s Relaxation delay: 4 s Total spin‐echo delay: 80 ms |
|||
|
EXO‐METABOLOME
Removal of the growing medium. Collect the medium in sterile condition making 1 mL aliquots. Store at −80 °C. |
Thaw at room temperature and shake. Add 350 μl of sample to 350 μL of sodium phosphate buffer (70 mm Na2HPO4; 20 % (v/v) 2H2O, 6.1 mm NaN3; 4.6 mm TMSP; pH 7.4). Transfer 600 μL of each mixture into a 5 mm NMR tube. |
Recommended magnetic field: 900 MHz Recommended acquisition temperature: 310 K 1D NOESY‐presat (Bruker: noesygppr1d) Scans: 32–64 Data points: 11 0060 Spectral width: 17 942.584 Hz Acquisition time: 3.07 s Relaxation delay: 4 s Mixing time: 0.01 s |
|
| TISSUES | Collect tissues in the most aseptic conditions possible. Cut tissue samples ≤0.5 cm in any single dimension. Snap freeze in liquid nitrogen within 30 min from collection. Stored in liquid nitrogen vapor. |
Trim frozen tissue samples (10–15 mg) to fit HR‐MAS ZrO2 rotor insert capacity. Fill the insert with 2H2O containing 5.8 mm TMSP. Cover the rotor inserts with plug and plug‐restraining screw and insert it into the 4 mm rotor for HR‐MAS.38 |
31, 38Recommended magnetic field: HR‐MAS 600 MHz Recommended acquisition temperature: 277 K 1D ZGPR Scans: 128–256 Data points: 32 768 Spectral width: 12 019.230 Hz Acquisition time: 1.36 s Relaxation delay: 2 s Rotor speed: 4 MHz 1D CPMG (Bruker: cpmgpr1d) Scans: 128–256 Data points: 32 768 Spectral width: 12 019.230 Hz Acquisition time: 3.07 s Relaxation delay: 1.36 s Total spin‐echo delay: 94 ms Rotor speed: 4 MHz |
[a] It is extremely important to: 1) minimize as much as possible the time between sample collection, processing and storage keeping the samples at 4 °C, if not differently specified; 2) use additive‐free tubes and laboratory materials to avoid sample contamination. [b] Minimize as much as possible the time between sample preparation and NMR spectral acquisition. [c] 1) Acquire NMR spectra in automatic mode using a refrigerated (4–6 °C) sample changer. 2) Acquire NMR spectra of samples in a totally random way, interleaving the samples of different groups to avoid batch effects. [d] According to Bruker guidelines.