Identification and Structural Characterization of Avian Beta-Defensin 2 Peptides from Pheasant and Quail

Pheasant and quail orthologs of Avian β-Defensin 2 (AvBD2) were identified by screening heterophil extracts using Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS), comparative profiling, chemical, and bioinformatic characterizations. In heterophil extracts of each pheasant and quail, we observed a single high intensity mass peak, corresponding to m/z 4114.8 and 4163.8, respectively which upon reduction and alkylation were shifted by a mass difference of 348 Da indicative of the modification of 3 internal disulfide bonds that exist in these peptides. The unmodified peptides, m/z 4114 and 4163, and their Carbamidomethylated (CAM) derivatives were purified by reverse phase HPLC. Using the purified peptides we determined their partial sequences by trypsin digestion followed by MALDI-TOF-MS, MALDI LIFT-TOF/TOF, and Edman degradation which showed their unambiguous homologies with other AvBD2. Combining all the results and aligning the sequence stretches with mature AvBD2 from other avian species, we deduced the amino acid sequences of pheasant and quail orthologs as, LFCKRGSCHFGRCPSHLIKVGSCFGFRSCCKWPWNA and LFCRRGTCHFGNCPSDQIKVGNCFGFRSCCRWPWDA, respectively. These sequence information were further confirmed by MALDI in-source decay (ISD) analyses. Both pheasant and quail AvBD2 peptides showed significant identities (>80%) with other known AvBD2 sequences.