The synthesis of individual fluorogenic substrates was carried out using classic solid phase peptide synthesis methods as described previously [28 (link)]. Each substrate was purified by HPLC on a Waters M600 solvent delivery module with a Waters M2489 detector system using a semi-preparative Waters Spherisorb S10ODS2 column. The solvent composition was as follows: phase A (water/0.1% TFA) and phase B (acetonitrile/0.1% TFA). The purity of each substrate was confirmed by analytical HPLC using a Waters Spherisorb S5ODS2 column. Finally, the molecular weight of each substrate was confirmed by mass spectrometry analysis. All compounds were at least 95% pure. Individual substrates were dissolved in 20 mM in DMSO and stored in −80°C until use.
Spherisorb s5ods2 column
Manufactured by Waters Corporation
Sourced in France
The Spherisorb S5ODS2 column is a reversed-phase high-performance liquid chromatography (HPLC) column. It is designed for the separation and analysis of a wide range of polar and non-polar compounds. The column features a 5 μm particle size and an octadecylsilane (ODS2) stationary phase, providing effective and reliable chromatographic performance.
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4 protocols using spherisorb s5ods2 column
1
Fluorogenic Substrate Synthesis and Purification
2
Phytochemical Profiling of Botanical Extracts
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The phytochemical characterization was performed in a Gilson apparatus equipped with a photodiode-array detector (PDA) (Gilson Electronics SA®, Villiers Le Bel, France) using a Spherisorb® S5 ODS-2 column (250 × 4.6 mm i.d., 5 µm) (Waters Corporation®, MA, USA) and a Nucleosil guard cartridge C18 (30 × 4 mm i.d., 5 µm) (Macherey-Nagel) at 24 °C. Data treatment was performed with the Unipoint software, version 2.10 (Gilson, WI, USA). For the mobile phase, a gradient of methanol and HPLC water–formic acid (95:5) was used: methanol 5–15% (0–10 min), methanol 15–30% (10–15 min), methanol 30–35% (15–25 min), methanol 35–50% (25–35 min), and methanol 50–80% (35–40 min), followed by an isocratic elution of methanol during 20 min at a flow rate of 1 mL/min. The volume of the sample injected was 100 µL. The UV–Visible spectra were acquired between 200 and 600 nm and the chromatographic profiles recorded at the wavelengths 280 and 320 nm.
3
Quantifying Phytomelatonin Levels Using HPLC
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A Jasco liquid chromatograph Serie-2000 (Tokyo, Japan) equipped with an online degasser, binary pump, auto sampler, thermo stated column and a Jasco FP-2020-Plus fluorescence detector were used to measure phytomelatonin levels. An excitation wavelength of 280 nm and an emission wavelength of 350 nm were selected. A Waters Spherisorb-S5 ODS2 column (250 × 4.6 mm) was used. The isocratic mobile phase consisted of water:acetonitrile (80:20) at a flow rate of 0.2 mL/min. The data were analyzed using the Jasco ChromNAV v.1.09.03 Data System Software. For correct identification, an in-line fluorescence spectral analysis (using the Jasco Spectra Manager Software) compared the excitation and emission spectra of standard melatonin with the corresponding peak of phytomelatonin in the samples [83 (link)].
4
HPLC Purification and Characterization of Peptides
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All chemical reagents were from commercial suppliers and used without further purification.
Reagents used for the solid-phase peptide synthesis are as follows Rink amide RA resin (particle size 200-300 mesh, loading 0.74 mmol/g), all Fmoc-amino acids, HBTU (O-benzotriazole- Individual substrates and ABPs were purified by HPLC on a Waters M600 solvent delivery module with a Waters M2489 detector system using a semi-preparative Waters Spherisorb S10ODS2 column. Solvent composition was as follows: phase A (water/0.1% TFA) and phase B (acetonitrile/0.1% TFA). The purity of each substrate and ABPs was confirmed with an analytical HPLC system using a Waters Spherisorb S5ODS2 column. The molecular weight of each substrate and ABP was confirmed by high-resolution mass spectrometry on a High Resolution Mass Spectrometer WATERS LCT premier XE with Electrospray Ionization (ESI)
and Time of Flight (TOF) module. The human 20S proteasome was purchased from Boston Biochem (Cambridge, MA, USA).
Reagents used for the solid-phase peptide synthesis are as follows Rink amide RA resin (particle size 200-300 mesh, loading 0.74 mmol/g), all Fmoc-amino acids, HBTU (O-benzotriazole- Individual substrates and ABPs were purified by HPLC on a Waters M600 solvent delivery module with a Waters M2489 detector system using a semi-preparative Waters Spherisorb S10ODS2 column. Solvent composition was as follows: phase A (water/0.1% TFA) and phase B (acetonitrile/0.1% TFA). The purity of each substrate and ABPs was confirmed with an analytical HPLC system using a Waters Spherisorb S5ODS2 column. The molecular weight of each substrate and ABP was confirmed by high-resolution mass spectrometry on a High Resolution Mass Spectrometer WATERS LCT premier XE with Electrospray Ionization (ESI)
and Time of Flight (TOF) module. The human 20S proteasome was purchased from Boston Biochem (Cambridge, MA, USA).
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