Tribute automated peptide synthesiser

Each peptide was chemically synthesised via solid-phase Fmoc chemistry using a Tribute automated peptide synthesiser (Protein Technologies, Tucson, AZ, USA), which was described in the previous study [61 (link)]. Rink amide resin was employed as solid phase for the synthesis process. The synthetic peptides were released from the resin by adding a cleavage cocktail, which contains 94% trifluoroacetic acid (TFA), 2% water, 2% thioanisole (TIS), and 2% 1,2-ethanedithiol (EDT) for 2 h at room temperature and further washed by diethyl ether three times. After lyophilisation, the crude peptides were then purified by RP–HPLC (Phenomenex Aeris PEPTIDE 5 μm XB-C18 column, 250 mm × 21.2 mm, Macclesfield, Cheshire, UK) by gradient elution from 90% solution A (99.95% dd H2O and 0.05% TFA) and 10% solution B (80% acetonitrile, 19.95% dd H2O and 0.05% TFA) to 100% solution B at a flow rate of 8 mL/min. The purity of peptides was identified by RP–HPLC and MALDI-TOF mass spectrometry (Voyager DE, Perspective Biosystem, Foster City, CA, USA) in positive detection mode using CHCA (α-cyano-4-hydroxycinnamic acid) as the matrix.

RNF and its analogues, RNF1 and RNF3L, were synthesised using standard Fmoc amino acids on a Tribute automated peptide synthesiser (Protein Technologies Inc., Tucson, AZ, USA). When the synthesis was completed, the peptides were cleaved from the resin and de-protected. Using a weak solution of hydrogen peroxide, the samples were oxidised to form the disulphide loop structure. After purification on an Adept CECIL4200 RP-HPLC (Amersham Biosciences Inc., Piscatawa, NJ, USA), Column: Phenomenex Aeris Peptide, C18, 250 mm × 10.0 mm (Phenomenex, Macclesfeld, UK), the masses of peptide samples were confirmed by using matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry (Perseptive Biosystems, Framingham, MA, USA). Alpha-cyano-4-hydroxycinnamic acid (CHCA) (Sigma Chemical Co., St. Louis, MO, USA) was used as matrix and was dissolved in TFA/H₂O/acetonitrile (0.05/49.95/50, v/v/v).