3 trimethylsilylpropionic acid tsp

Urine samples were stored at -80 °C until analysis [1 (link)]. Samples were filtered to remove protein with Centrifree Ultrafiltration devices (Millipore, Bilerica, MA). Filters were washed 5 times to remove glycerol. Samples were filtered by centrifugation at 6000 rpm for 2–3 h at 4 °C. For NMR analysis, 1 mL of filtrate was mixed with 0.5 mL of 0.2 M sodium phosphate buffer. The solution was placed on ice for 10 min and then centrifuged at 7000 g at room temperature for 10 min. 500 μL of the supernatant was withdrawn and combined with 50 μL of 1 mM 3-(trimethylsilyl) propionic acid (TSP, Sigma-Aldrich, St. Louis, MO, USA) [9 ]. The pH of the solution was measured, then the solution was transferred to 5 mm NMR tubes for analysis.

The palustrin-Ca peptide (MW = 3304 g/mol, purity > 95%) was purchased from ProteoGenix (Paris). The identity and purity of the peptide were confirmed using mass spectrometry and high performance liquid chromatography (Supplementary Information). 4.5 g of the peptide was dissolved in 0.6 mL of a 50% (v/v) TFE- $d_3$ /H₂O solution, resulting in a final peptide concentration of 2.27 mM. 3-trimethylsilyl propionic acid (TSP) and 2,2,2-trifluoroethanol (TFE- $d_3$ ) of analytical grade were obtained from Sigma-Aldrich (Ireland).

The crude drug form of VB was purchased from Jiangsu Medicine Company (Nanjing, China) and authenticated by Prof. Ming-Jian Qin (Department of Medicinal Plants, China Pharmaceutical University). 3-Trimethylsilylpropionic acid (TSP) was obtained from Sigma (St. Louis, MO, USA). Deuterium oxide (D₂O, 99.9%) was purchased from Sea Sky Bio Technology Co. Ltd (Beijing, China). Pentobarbital sodium was obtained from Fuoshan Chemical Factory (Fuoshan, China). Ultra-pure distilled water, prepared using a Milli-Q purification system, was utilized in the experiments.