Direct screening identifies mature beta-defensin 2 in avian heterophils

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 to 20 kDa with an objective to identify specific 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 m/z 4129.6 for turkey. 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. A 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. Both AvBD2 peptides are 36 amino acids long including a highly conserved region with 6 invariant cysteines forming the 3 disulfide bonds characteristic of defensins. The method confirmed the existence of the complete mature peptide sequence of the turkey heterophilic BD2 previously proposed based on cDNA analysis. These results demonstrate that screening of the crude extract by MALDI-TOF-MS can identify cell- or tissue-associated peptides in their functional or mature forms, raising the possibility that such peptides can be used as biomarkers in their altered physiological states.