The peptidomics was conducted using the ultra-high performance liquid chromatography tandem mass as described previously (26 (link)) with some modifications. In brief, the peptide extract (4 mg) was dissolved in 0.1% trifluoroacetic acid and was then desalted with the Oasis HLB column (Waters, Milford, MA, USA). The eluents were dissolved by 50% acetonitrile with 0.5% trifluoroacetic acid and were desalted with the Oasis MCX column (Waters, Milford, MA, USA). After being freeze-dried, the samples were dissolved at 10 μg/μl using 2% acetonitrile and 0.1% formic acid. The samples (10 μl) were analyzed using the Easy-nLC 1200 UPLC-Q Exactive HF-X tandem mass spectrometer (Thermo, Waltham, MA, USA), coupled with a Thermo C-18 column (Thermo, Waltham, MA, USA). The instrument was operated in a single charge mode ([M+H]+) at the range of 350–1,500 (m/z). The peptide sequences were identified using Proteome DiscovererTM version 2.4 (Thermo, Waltham, MA, USA) according to the protein sequences of Siniperca chuatsi. False discovery rate <0.01, unique peptide ≥1, and Sequest HT >0 were used as the criteria to confirm the credible peptides. The peptides with ACE inhibitory activity from Chouguiyu were further predicted using AHTpin (http://crdd.osdd.net/raghava/ahtpin/ ).
Thermo c18 column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Thermo C18 column is a reversed-phase liquid chromatography (RPLC) column used for the separation and analysis of a wide range of organic compounds. It features a chemically bonded octadecylsilane (C18) stationary phase, which provides high-resolution separation and excellent retention of non-polar and moderately polar analytes. The Thermo C18 column is designed for use in a variety of RPLC applications, including pharmaceuticals, environmental analysis, and food and beverage testing.
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Lab products found in correlation
Oasis hlb column, by Waters Corporation (1 mentions)
Oasis mcx column, by Waters Corporation (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Tsq quantum access max, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 system, by Thermo Fisher Scientific (1 mentions)
Perkin elmer series 200, by PerkinElmer (1 mentions)
4 protocols using thermo c18 column
1
Peptidomics Analysis of Chouguiyu Protein
2
Quantitative Analysis of Lacidipine in Plasma
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The lacidipine plasma concentrations were determined by a validated ultra performance liquid chromatography-dual mass spectrometry (UPLC-MS/MS) method after liquid–liquid extraction by tert-butyl methyl ether with nimodipine as internal standard. The chromatographic separations were acquired on an ACQUITY UPLC™ system (Waters Corp., Milford, MA, USA) and Thermo C18 column (50 mm × 2.1 mm, 2.6 µm; Thermo Fisher Scientific, USA) with a mobile phase composed of acetonitrile and water containing 0.1% formic acid (83:17; v/v) at a flow rate of 0.2 ml/min. The quantitation was performed by Waters Tandem Quadrupole (TQ) Detector (Waters). The mass spectrometer was operated with electrospray ionization (ESI) source in positive ionization mode and the compounds were analyzed by multiple reaction monitoring (MRM) of the transitions of m/z 473.47→354.28 for lacidipine and m/z 419.25→343.18 for nimodipine, respectively.
3
Quantitative Analysis of Analytes via LC-MS/MS
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The analysis utilized an UltiMate 3000 system connected to a TSQ Quantum Access Max triple-stage quadropole mass spectrometer (Thermo Fisher Scientific). Chromatographic separation was achieved using a Thermo C18 column (100 × 2.1 mm, 1.8 µm; Thermo Fisher Scientific), and the column temperature was maintained at 25 °C.
The mobile phases consisted of 0.1 % formic acid in distilled water (A) and acetonitrile (B). The gradient elution method employed was as follows: 95 % A at 0–2 min, 95–70 % A at 2–4 min, 70–60 % A at 4–7 min, 60–20 % A at 7–10 min, 20–5 % A at 10–13 min, 5 % A at 13–15 min, 5–50 % A at 15–17 min, and 50–95 % A at 17–20 min. The flow rate was set at 0.3 mL/min, with a 5 µL. Mass spectrometry was conducted in both positive and negative ion modes, and the MS spectra were acquired in multiple reaction monitoring (MRM) mode. The instrumental parameters were as follows: vaporizer temperature, 350 °C; capillary temperature, 300 °C; spray voltage, 3000/2500 V; shealth gas pressure at 35 Arb; and aux gas pressure, 10 Arb. Quantitative parameters are detailed inTable S2 .
The mobile phases consisted of 0.1 % formic acid in distilled water (A) and acetonitrile (B). The gradient elution method employed was as follows: 95 % A at 0–2 min, 95–70 % A at 2–4 min, 70–60 % A at 4–7 min, 60–20 % A at 7–10 min, 20–5 % A at 10–13 min, 5 % A at 13–15 min, 5–50 % A at 15–17 min, and 50–95 % A at 17–20 min. The flow rate was set at 0.3 mL/min, with a 5 µL. Mass spectrometry was conducted in both positive and negative ion modes, and the MS spectra were acquired in multiple reaction monitoring (MRM) mode. The instrumental parameters were as follows: vaporizer temperature, 350 °C; capillary temperature, 300 °C; spray voltage, 3000/2500 V; shealth gas pressure at 35 Arb; and aux gas pressure, 10 Arb. Quantitative parameters are detailed in
4
Purification of S. ricini Peptides
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S. ricini, hydrolyzed with trypsin, was purified by RT-HPLC (Perkin Elmer 200 Series, PerkinElmer Inc., Waltham, MA, USA) according to the method of Xing, L.-j., et al. [14 (link)] with a slight modification. Purification was carried out using a Thermo C18 column (diameter 4.5 mm, length 250 mm, particle size 5 µm, Thermo Fisher Scientific Inc., Waltham, MA, USA). For separation part, solvent composition of the mobile phase at the two channels was 0.1% (v/v) formic acid and 2% (v/v) acetonitrile for channel A and 0.1% (v/v) formic acid and 100% (v/v) acetonitrile for channel B. The peptides were separated by a gradient elution to 30% B in 2 min, to 80% B in 18 min, to 100% B in 7 min.
The flow rate was set at 0.3 mL/min, measured at wavelengths of 220 nm, and collected fractions were stored at −20 °C for further analysis.
The flow rate was set at 0.3 mL/min, measured at wavelengths of 220 nm, and collected fractions were stored at −20 °C for further analysis.
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