Stapled peptide synthesis and purification

Unmodified and hydrocarbon stapled peptides were synthesized by Fmoc-based solid phase peptide synthesis and purified by reverse-phase HPLC with a C18 column (Agilent, Palo Alto, CA) as previously reported³⁹. The first series of RFP peptides (RFP1-RFP13) were analyzed by LC/MS using a C18 reverse-phase column (Agilent, 2.1 × 150 mm, pore size 80 Å, particle size 3.5 µM); Buffer A (H₂O/0.1% TFA) and Buffer B (ACN/0.1% TFA); and a 15 min method with the following gradient (flow rate 0.5 mL/min): 10–100% buffer B over 10 min, 100% buffer B for 2 min, 100–10% buffer B over 1 min, and 10% buffer B over 2 min. Optimized RFP peptides (denoted by * on retention time in Supplementary Table ³) were analyzed by LC/MS using a C18 reverse-phase column (Phenomenex, 5.0 × 50 mm, pore size 110 Å, particle size 5 µm); Buffer A (5/95/0.1% ACN/H₂O/TFA) and Buffer B (95:5:0.1% ACN/H₂O/TFA); and a 20 min method with the following gradient (flow rate 0.5 mL/min): 0% buffer B over 3 min, 0–65% buffer B over 15 min, 65–100% buffer B over 1 min; 100–0% buffer B over 1 min. Purified peptides were lyophilized, quantified by A₂₈₀, dissolved in DMSO as 10 mM stocks and stored at −20 °C.