Hplc labsolution

Small humanin-like peptide 1 to 6 (SHLP1-6) were purchased from AnyGen [http://www.anygen.com/eng/]. For each peptide, the Fmoc-Ser (tBu)-Wang resin was allowed to swell in dimethylformamide (DMF). The amino acid was then deprotected using a solution of 20% piperidine in DMF. Coupling was carried out using Hexafluorophosphate Benzotriazole Tetramethyl Uronium (HBTU), N-methylmorpholine (NMM) in DMF. Stepwise deprotection and coupling of amino acids was repeated until the desired peptides was synthesized. The peptides were cleaved from dried resin using a trifluoroacetic acid (TFA) solution containing 2.5% 1, 2 ethanedithiol (EDT), 5% thioanisole and 5% distilled water. Crude peptides were precipitated in ether, and dried under vacumm. Peptides were purified using commercial columns (YMC-Triart C18/S-5 μm/12 nm.5μm (20 x 250 mm) and each fraction was obtained at a flow rate of 1 mL/min. The collected fractions were analyzed via HPLC (Shimadzu HPLC LabSolution) and MALDI-TOF MS (AXIMA Assurance, Shimadzu). The fractions containing the pure peptide were mixed and then lyophilized. SHLP2 were freshly prepared at 1 µg/µL by diluting in saline, and were freshly used for mice. The information of SHLP peptide sequence used in study were listed in Supplementary Table 3.

The transcriptome database of O. minor was screened for AMPs, and the prohibitin-2 cDNA sequence was considered a candidate for designing a novel AMP. The prohibitin-2 cDNA sequence was submitted to the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/, accessed on 7 January 2022) database with accession number MW939430. To design octoprohibitin, the C-terminal region of the prohibitin-2 AA sequence was selected as a template. The novel AMP, octoprohibitin (WRVCKQSEKVILNPFWKKKKKKKFRV), which contains 26 AA residues, was synthesized by a solid-phase peptide synthesis technique (AnyGen Co., Gwangju, Korea), and purified by reverse-phase high-performance liquid chromatography using a SHIMADZU C18 analytical column (Shimadzu HPLC LabSolution, Kyoto, Japan). Mass analysis was conducted using linear MALDI-TOF mass spectrometry (AXIMA Assurance, MALDI-TOF; Shimadzu, Kyoto, Japan). The three-dimensional structure of octoprohibitin was generated using APPTEST (https://research.timmons.eu/apptest, accessed on 7 January 2022), and the images were visualized using Discovery Studio Visualizer (Version 21.1.0.20298, Biovia, CA, USA). The helical wheel projection of octoprohibitin was derived by Netwheel, Peptides Helical Wheel, and Net projection maker (http://lbqp.unb.br/etWheels/, accessed on 7 January 2022) [18 (link)].