Submitted to: Poultry Science
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/21/2008
Publication Date: 2/1/2009
Citation: Kannan, L., Rath, N.C., Liyanage, R., Lay, J.O. 2009. Direct screening identifies mature beta-defensin 2 in avian heterophils. Poultry Science. 88(2):372-379. Interpretive Summary: Peptides are small proteins which play important roles in regulating cell’s functions such as growth, survival and communication. We developed a strategy using a method called ‘mass spectrometry’ to identify some of these peptides in the cells of interest. Using a poultry blood cell type called heterophils which fights against pathogens we identified an antimicrobial peptide named beta-defensin 2 in chickens and turkeys. It appears that mass spectrometry method may be useful to identify cell or tissue specific peptides or their disease induced changes.
Technical Abstract: Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to screen avian heterophils in the m/z range of 1-20 kDa with an objective to identify the cell associated peptides that may be reflective of their functional physiology. The MALDI-TOF-MS profiles of crude heterophil extract showed a high intensity peak with average mass of m/z 3916.1 for chicken and at m/z 4129.6 for turkey respectively. To identify these peaks, we first purified m/z 3916.1 from chicken bone marrow extract using reverse phase high performance liquid chromatography (RP-HPLC). Edman sequencing and peptide mass fingerprinting exclusively confirmed this peptide as beta-defensin 2 (BD2) or gallinacin-2, a broad range antimicrobial peptide. Uniprot database search followed by the MASCOT sequence query revealed m/z 4129.6 to be the corresponding turkey ortholog of avian'beta-defensin 2 (AvBD2), also called turkey heterophil peptide 2 (THP2). Both AvBD2 peptides are 36 amino acids long including a highly conserved region with 6 invariant cysteines forming three disulfide bonds characteristic of defensins. These results demonstrate that screening of the crude extract by MALDI-TOF-MS can identify cell or tissue associated peptides in their functional/mature forms. This study also confirms the existence and the complete mature peptide sequence of the turkey heterophilic BD2 previously proposed based on cDNA analysis.