Determining the cleavage site for the mature antimicrobial peptide of Nile tilapia β-defensin using 2D electrophoresis, western blot, and mass spectrometry analysis. Issue 62 (March 2017)

• Record Type: Journal Article
• Authors: Chang, Chin-I; Chen, Li-Hao; Hu, Yeh-Fang; Wu, Chia-Che; Tsai, Jyh-Ming
• Is Part Of: Fish & shellfish immunology. Issue 62(2017)
• Page Start: 41
• Page End: 46
• Publication Date: 2017-03
• Subjects: β-defensin -- Cleavage site -- Oreochromis niloticus -- Antimicrobial peptide; Fishes -- Immunology -- Periodicals; Shellfish -- Immunology -- Periodicals; Poissons -- Immunologie -- Périodiques; Crustacés -- Immunologie -- Périodiques; 571.9617
• DOI: 10.1016/j.fsi.2017.01.010
• Languages: English
• ISSNs: 1050-4648

Abstract

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 ( 25 ASFPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL 66 ) 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 ( 27 FPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL 66 ) 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. Highlights: The cleavage site of mature Nile tilapia β-defensin was confirmed. Computer-predicted β-defensin was synthesized to produce Ab for Western blotting. The spot of β-defensin was recognized and extracted from the 2-DE gel. The precise molecular mass of this spot was determined by LC-MS/MS spectrometry. The calculated mass of a sequence was completely matched the LC-MS/MS result.