3.13. Preparation of NMR Samples from Serum

Thaw the serum samples stored at −80 ^oC on ice.

500 uL of serum sample is transferred into a new Eppendorf tube and mixed with 1 mL of methanol to precipitate proteins.

Samples are centrifuged at 15,000 × g for 10 min at 4 ^oC.

Transfer the supernatants into new microcentrifuge tubes.

Remove the methanol by rotary evaporation using a SpeedVac.

The samples are flash-frozen in liquid nitrogen and then dried by lyophilization.

The dried samples are reconstituted into 550 μL of 50 mM phosphate buffer in 99.8% D₂O at pH 7.2 (uncorrected, see Note 3). 50 μM (for 1D ¹H NMR) or 500 μM (for 2D NMR) TMSP-d₄ are added to each sample as a chemical shift and concentration reference.

The sample is transferred to a 4” 5 mM SampleJet NMR tube with a RAININ XLS pipette (see Note 12).

Repeat steps 1 to 8 for each replicate and for each group (see Note 9).

Each sample is added to a 96 well plate SampleJet configuration equilibrated to 4 °C to prevent metabolite degradation (see Figure 5).