Ultimate 3000 semiprep hplc

Peptides were produced on solid phase (Fmoc-Rink Amide MBHA, capacity = 0.67 mmol/g) resin in an automated peptide synthesizer (Syro-I, Biotage, Uppsala, Sweden) using standard Fmoc/tBu strategy with DIC/HOBt coupling reagents. Cf was coupled to the N-terminus of the peptides by using DIC/HOBt coupling method. Peptides were cleaved from the resin with TFA/H₂O/TIS (9.5:2.5:2.5, v/v) mixture (2 h, RT). After filtration, compounds were precipitated in cold diethyl ether, centrifuged (4,000 rpm, 5 min) and freeze-dried from water.
RP-HPLC purification was performed on an UltiMate 3000 Semiprep HPLC (Thermo Fisher Scientific) with a Phenomenex Jupiter Proteo C-12 column (250 × 10 mm) using gradient elution, consisting of 0.1% TFA in water (eluent A) and 0.1% TFA in acetonitrile/water = 80/20 (v/v) (eluent B).
Purified peptides were analyzed by LC-MS using a Thermo Scientific Q Exactive Focus Hybrid Quadrupole-Orbitrap Mass Spectrometer. For the separation, a Waters Acquity UPLC BEH C18 (1.7 µm, 150 × 2.1 mm) column was used with a flow rate of 0.3 ml/min.
Residual TFA and TFA counter-ion was removed by using an acetate-exchange resin. For the detailed description, see Supplementary Information 1.1).

The purification of crude peptides and conjugates was performed on an UltiMate 3000 Semiprep HPLC (Thermo Fisher Scientific, Waltham, MA, USA) with a Phenomenex Jupiter Proteo C-12 column (250 × 10 mm) using gradient elution, consisting of 0.1% TFA in water (eluent A) and 0.1% TFA in acetonitrile/water = 80/20 (v/v) (eluent B).
The crude and purified peptides were analyzed by analytical RP-HPLC (Shimadzu prominence HPLC system) with a Phenomenex Jupiter Proteo C-12 column (150 × 4.6 mm) using gradient elution, consisting of 0.1% TFA in water (eluent A) and 0.1% TFA in acetonitrile/water = 80/20 (v/v) (eluent B).

Peptides were prepared on TGS Wang resin (250 mg, capacity = 0.27 mmol/g) in an automated peptide synthesizer (Syro-I, Biotage, Uppsala, Sweden) using Fmoc/tBu strategy with DIC/HOBt coupling reagents. Palmitic acid was coupled on the N-terminus of the peptides using DIC/HOBt coupling reagents. After the synthesis was complete, peptides were cleaved from the resin with TFA in the presence of scavengers (H₂O and TIS, 3–3 v/v%). Crude products were precipitated in cold diethyl ether, centrifuged (4000 rpm, 5 min), and lyophilized from H₂O/AcN. Palmitoylated peptides were then purified by RP-HPLC on a Phenomenex Jupiter Proteo C12 column (10 μm, 90 Å, 10 mm × 250 mm) with linear gradient elution using 0.1% TFA in H₂O (eluent A) and 0.1% TFA in AcN:H₂O = 80:20 (v/v) (eluent B) on an UltiMate 3000 Semiprep HPLC (Thermo Fisher Scientific, Waltham, MA, USA). Purified peptides were analyzed by RP-HPLC using an LC-40 HPLC System (Shimadzu, Kyoto, Japan) on an analytical Phenomenex Jupiter Proteo C12 column (10 μm, 90 Å, 4.6 mm × 150 mm). The flow rate was 1 mL/min, and the gradient was 5–100 B% in 20 min (UV detection at λ = 220 nm). High-resolution mass spectra were acquired by direct injection to a Thermo Scientific QExactive Focus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Waltham, MA, USA). Data were analyzed by Xcalibur program (Thermo Fisher Scientific, Waltham, MA, USA).