L 3000 hplc system

First dimension RP separation was performed on an L-3000 HPLC system (Rigol) using a Durashell RP column (5 μm, 150 Å, 250 × 4.6 mm internal diameter; Agela). Mobile phase A (2% acetonitrile, pH 10.0) and mobile phase B (98% acetonitrile, pH 10.0) were used for RP gradient. Dried peptides were resuspended in 200 μl mobile phase A. The solvent gradient was set as follows: 5–8% B, 2 min; 8–18% B, 11 min; 18–32% B, 9 min; 32–95% B, 1 min; 95% B, 1 min; 95–5% B, 2 min. Tryptic peptides were separated at an eluent flow rate of 1.5 ml/min and monitored at 214 nm. Dried samples were reconstituted in 15 μl of 0.1% (v/v) formic acid and 2% (v/v) acetonitrile in water for subsequent analyses.

The pooled mixture from labeled samples was dissolved in mobile phases A and fractioned by Durashell RP column (5 µm, 150 Å, 250 mm × 4.6 mm, Agela) from L-3000 HPLC system (Rigol, China). Eluent was collected every minute, pooled into 12 samples and dried under vacuum. Peptides were eluted from the C18 analytical column with a 40 min gradient at a speed of 350 nl/min on an Eksigent Ultra HPLC (AB sciex, USA). The mass spectrum conditions for Triple TOF 5600 was set as follow: The spray voltage was set at 2.5 kV and the temperature of heater was 150 °C. The mass spectrum scan range was m/z 350–1250 and the tandem mass spectrometry (MS/MS) scan range was m/z 100–1500.

The co-precipitated proteins in Co-IP assay were subjected to mass spectrometry analysis using the L-3000 HPLC System (RIGOL, Beijing, China) after desalination. Raw mass spectrometry data were processed by Proteome Discoverer2.4 (Thermo Fisher Scientific).

Mobile phases A (2% acetonitrile, adjusted pH to 10.0 using ammonium hydroxide) and B (98% acetonitrile) were used to develop a gradient elution. The lyophilized powder was dissolved in solution A and centrifuged at 12,000 × g for 10 minutes at room temperature. The sample was fractionated using a C18 column (Waters, Milford, United States of America BEH C18, 4.6 × 250 mm, 5 μm) on a Rigol, Portland, United States of America L3000 HPLC system, and the column oven was set as 45°C. The detail of elution gradient is shown in Supplementary Table S1. The eluates were monitored at UV 214 nm, collected in a tube per minute, and combined into 10 fractions finally. All fractions were dried under vacuum and then reconstituted in 0.1% (v/v) formic acid in water.

Based on the screening of active markers, we selected 9 bufadienolide standards as quality evaluation indicators for Bufonis Venenum. Samples of Bufonis Venenum from different batches (Sample information is shown in Table 1) and from different production areas were collected. Each sample was ultrasonically extracted with 90% methanol for 1 h and the supernatant was centrifuged at high speed for liquid phase measurement. Quantitative detection of the samples was performed on an L-3000 HPLC system (RIGOL) using an X-Charge column (4.6 mm × 250 mm, 5 µm). A gradient elution of solvent A (0.1% formic acid in water) and solvent B (acetonitrile) was applied as follows: 0–4 min, 20–38% B; 4–20 min, 38% B; 20–30 min, 38–90% B; 30–34 min, 90–90% B; 34–35 min, 90–20% B; 35–45 min, 20% B. The column temperature was 35 °C; volume flow was 0.7 mL/min; and the injection amount was 20 μL, detected at a wavelength of 296 nm.

The free amino acid content was determined by a previously described pre-column derivatization RP-HPLC method [95 (link)], with a slight modifications. For hydrolysis, approximately 0.1 g of sample was mixed with hydrochloric acid (0.1 M, 1 mL) and vigorously vortexed for 60 s, after which the sample was left to stand at 4 °C overnight. Then, the tube contents were centrifuged, and the supernatant was membrane-filtered to obtain the hydrolysate. For derivatization, a standard amino acid solution or sample hydrolysate (200 μL) was placed into a 2 mL tube and 20 μL of internal standard dl-norleucine solution was added. Next, 100 μL of triethylamine-acetonitrile (2.8:17.2, v/v) and 100 μL of phenylisothiocyanate-acetonitrile (0.125:10, v/v) were added to each tube, mixed and incubated for 1 h at 25 °C. Next, 400 μL of n-hexane was added to each tube, mixed and placed at 25 °C for 10 min. The resulting lower liquid was filtered and then used for detection. Chromatographic analysis was carried out using a RIGOL L-3000 HPLC system. The Amethyst C18-H column (250 mm × 4.6 mm, 5 μm) was maintained at 40 °C. The flow rate was set at 1.0 mL min⁻¹ and the injection volume was 10 μL. The measurements were obtained at a wavelength of 254 nm.

Prepared mobile phase A liquid (2% acetonitrile, 98% water, ammonia water adjusted to pH = 10) and B liquid (98% acetonitrile, 2% water, ammonia water adjusted to pH = 10). Used 150μl of solution A to dissolve the lyophilized powder of the sample, centrifuged at 12000g for 10min at room temperature, and took the supernatant for injection. The TMT labelled peptide mixture was fractionated by a Durashell column from Agela (4.6mm×250mm i.d, C18, 5μm) by L-3000 HPLC system (Rigol, China). A total of 100 tubes were collected with the speed of 1 tube per minute, and finally combined into 10 fractions. All fractions were dried by a rotary vacuum concentrator.