title: Determining the cleavage site for the mature antimicrobial peptide of Nile tilapia β-defensin using 2D electrophoresis, western blot, and mass spectrometry analysis.
abstract: Several proteomic techniques were used to determine the cleavage site of the mature antimicrobial peptide of Nile tilapia β-defensin. The computer-predicted Nile tilapia β-defensin (25ASFPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL66) composed of 42 amino acids was chemically synthesized and prepared to produce an antibody for Western blotting. Total proteins from the skin of the Nile tilapia were separated on two-dimensional electrophoresis, and the spot of Nile tilapia β-defensin was recognized using Western blot analysis. It was then excised and extracted from the gel. The precise molecular mass of this spot was determined by LC-MS/MS spectrometry. Four major peptides were discovered, with molecular weights of 4293.2 Da, 4306.5 Da, 4678.9 Da, and 4715.0 Da. The calculated mass of the 40-amino-acid sequence (27FPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL66) of Nile tilapia β-defensin starting from Phe27 and ending with Leu66 was 4293.18 Da, which completely matched the 4293.2 Da peptide that was obtained from the mass spectrometry analysis. This result confirmed that the cleavage site for the mature C-terminal Nile tilapia β-defensin is at residue Ser26-Phe27, not at Ala24-25 as predicted by computer analysis. This study provides a simple but reliable model to determine the cleavage site for a mature antimicrobial peptide.
authors.affiliation: Aquaculture Division, Fisheries Research Institute, Council of Agriculture, 199 Hou-Ih Road, Keelung 20246, Taiwan. Electronic address: cichang@mail.tfrin.gov.tw.
authors.forename: Chin-I
authors.initials: CI
authors.lastname: Chang
authors.affiliation: Aquaculture Division, Fisheries Research Institute, Council of Agriculture, 199 Hou-Ih Road, Keelung 20246, Taiwan.
authors.forename: Li-Hao
authors.initials: LH
authors.lastname: Chen
authors.affiliation: Aquaculture Division, Fisheries Research Institute, Council of Agriculture, 199 Hou-Ih Road, Keelung 20246, Taiwan.
authors.forename: Yeh-Fang
authors.initials: YF
authors.lastname: Hu
authors.affiliation: Aquaculture Division, Fisheries Research Institute, Council of Agriculture, 199 Hou-Ih Road, Keelung 20246, Taiwan.
authors.forename: Chia-Che
authors.initials: CC
authors.lastname: Wu
authors.affiliation: Department of Marine Biotechnology, National Kaohsiung Marine University, 142 Hai-Chuan Road, Kaohsiung 81157, Taiwan.
authors.forename: Jyh-Ming
authors.initials: JM
authors.lastname: Tsai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018407:Bacterial Physiological Phenomena; D015153:Blotting, Western / Q000662:veterinary; D002853:Chromatography, Liquid / Q000662:veterinary; D023681:Cichlids / Q000235:genetics* / Q000276:immunology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000662:veterinary; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D040901:Proteomics; D016415:Sequence Alignment / Q000662:veterinary; D053719:Tandem Mass Spectrometry / Q000662:veterinary; D023083:beta-Defensins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D023181:Antimicrobial Cationic Peptides; D029941:Fish Proteins; D023083:beta-Defensins
keywords: Antimicrobial peptide; Cleavage site; Oreochromis niloticus; β-defensin