Conflicts with diarrheal pathogens trigger rapid evolution of an intestinal signaling axis

Clayton M. Carey; Sarah E. Apple; Zoe A. Hilbert; Michael S. Kay; Nels C. Elde

To generate a C-terminal acid with the last amino acid of the sequence pre-loaded and a density of ~0. Oxidation of free cysteine residues (STp, STh, Y-ST, V-ST) Oxidation of free cysteine residues in purified peptides was accomplished using air oxidation of solid peptide dissolved in peptide oxidation buffer (50 mM Tris Base, 2% DMSO, pH 8 at 37°C) at ≤0.4 mg/mL (to ensure intramolecular disulfide formation) for ~17 h at 37°C with shaking at 200 rpm. The reaction was quenched with glacial acetic acid (final concentration of 5%), and the pH was confirmed to be between pH 3-4. Reaction solution was then diluted 2-fold with HPLC Buffer A, spun for 10 min at 4696 xg and purified by reverse phase HPLC on a Phenomenex Jupiter 4-µm Proteo C12 column using a gradient of 36-40 % Buffer B (V-ST), 24 to 29% Buffer B (Y-STa), 30-35% Buffer B (STp), 25.5-30% Buffer B (STh) over 35 min with a flow rate of 10 mL/min. All HPLC methods started at 20% Buffer B to prevent peptide precipitation. Fractions were collected and checked for purity by LC/MS (10-90 or 10-70% Buffer B over 10 min) and lyophilized. For each peptide, eight distinct peaks were collected ( Figure S5A ) and tested for activity in the cellular cGMP assay. The most active peaks were: oxidized STp peak #2, oxidized STh peak #8, oxidized V-ST peak #2, and oxidized Y-ST peak #1 ( Figure S5B and C) .