Automated Peptide Synthesis and Purification

Peptides were synthesized using a PS3 automated peptide synthesizer (Protein Technologies, Phoenix, AZ) via standard Fmoc/tbu solid-phase peptide synthesis unless noted in Table 1. Manual coupling of amino acids was performed by incubating the peptide resin in a solution of amino acid (4 eq.) and HCTU (3.9 eq.) in 0.4 M N-methylmorpholine in DMF for 3 h. Fmoc deprotection was performed by two 30-min incubations in 20% (v/v) piperidine in DMF. Biotinylated resin was prepared from NovaPEG rink amide resin as reported previously^{27 (link)} and was used for the synthesis of all biotinylated peptides. Peptides were cleaved off the resin by incubating in a cleavage cocktail containing TFA/DMB/TIPS/EDT (90:5:2.5:2.5 v/v/v/v) for 2.5 h followed by double precipitation in cold ether. EDT was included in the cleavage cocktail only for peptides containing a free cysteine after cleavage. The crude peptides were purified by RP-HPLC (Agilent 1200, Santa Clara, CA) to 95% purity using a Phenomenex Fusion-RP C18 semi-preparative column (Torrance, CA) at the flow rate of 5 mL/min with H₂O (0.1% TFA) and ACN (0.1% TFA) as a mobile phase. Molecular weights of the purified peptides were confirmed by MALDI-ToF Mass Spectrometry (MS).