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

Crude peptides were first validated by LC/MS and analytical HPLC, as described above. Oxidation of free cysteine residues was accomplished using air oxidation of solid peptide dissolved in peptide oxidation buffer at ≤0.4 mg/mL for 14-18 h at 37°C with shaking at 200 rpm. The reaction was quenched with glacial acetic acid (5% final concentration) and the pH was confirmed to be between 3-4. Reaction solution was then diluted 2-fold with HPLC Buffer A, spun for 10 min at 4696 xg and purified by reversephase HPLC on a Phenomenex Jupiter 4-µm Proteo C12 column (see specs above) using a gradient Dry purified peptides with a single formed disulfide and Cys(Acm) protecting groups at positions C7 and C15 (1-5 mg) were dissolved in Buffer A to a concentration of ~400 µM. Fresh I2-TFA mixture was prepared (~10 mg I2 dissolved in 5 mL ACN, then added to a solution of 15 mL H2O with 0.6 mL TFA), and the I2-TFA mix was added (2x volume) to the peptide-Buffer A solution. This mixture reacted on a rotator at RT for 20 min before quenching with 1 M ascorbic acid added drop-wise until solution changes from rust color to colorless, clear solution (typically less than 30 μL). Quenched solution was diluted with one volume Buffer A, spun 10 min at 4696 xg, and transferred to a new tube for purification. Same-day purification proceeded via reverse-phase HPLC using a Phenomenex 5-μm C18-Kinetex column C10 100Å (150 x 4.6 mm) with a gradient of 20-52% Buffer B (human uroguanylin), 14-23% Buffer B (P. vampyrus), 10-30% Buffer B (M. lucifugus), or 22-30% Buffer B (E. fuscus) over 30 min at 2 mL/min to achieve baseline separation of peptide topoisomers. Two individual peaks were collected separately and their mass confirmed by LC/MS. Generally the larger peak was determined to be the active isomer (consistent with previous studies 24 ). The uroguanylin topoisomers were found to interconvert at RT (as seen previously for human uroguanylin [24] [25] [26] , with M. lucifigus converting the fastest-within 30 min of peak purification). Therefore, fractions were placed on ice immediately after purification until confirmation by LC/MS, then pooled and lyophilized. All dry, purified peptides were stored in parafilmed containers in the dark at -20°C. We tested the order of disulfide bond formation and found that forming the C7-C15 disulfide second yielded more of the active isomer compared to forming the C4-C12 disulfide second.