α cyano 4 hydroxycinnamic acid

Proton channel peptides, shown in Fig. 2 and Supplementary Table S1, were synthesized using Fmoc-based solid-phase peptide synthesis on either a Biotage Initiator Alstra microwave synthesizer or a Syro II parallel peptide synthesizer. All peptides were synthesized with a free amine N-terminus and a C-terminal carboxamide using Tentagel S-RAM resin (Chem-Impex Int’l Inc) with 0.22–0.24 mmol/g. Following synthesis, the peptides were cleaved from the resin with a trifluoroacetic acid: triisopropylsilane: water (TFA:TIPS:H₂O, 95:2.5:2.5) solution, precipitated out with cold diethyl ether, redissolved in 50% 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and water, and purified using reverse phase HPLC on a C4 prep column (Vydac) with a gradient of solvents A (water, 0.1% TFA) and B (isopropanol:acetonitrile:water:TFA, 60:30:9.9:0.1) at a flow rate of 10 mL/min. Peptides were then lyophilized until dry. Following successful purification, peptides were confirmed to be >90% pure with analytical HPLC over a C4 column (Phenomenex) and confirmed to have the correct mass with MALDI mass spectrometry (Shimadzu AXIMA Performance) using α-cyano-4-hydroxycinnamic acid (Sigma) as the matrix (Supplementary Table S1). Peptides were then dissolved in ethanol to the appropriate stock concentrations and subsequently used for all experiments as described.