L.N.F. Darville, M.E. Merchant, A. Hasan, K.K. Murray, “Proteome analysis of the leukocytes from the American alligator (Alligator mississippiensis) using mass spectrometry,” Comp. Biochem. Physiol. Part D Genomics Proteomics. 5 (2010) 308โ€“316. doi:10.1016/j.cbd.2010.09.001.

Abstract: Mass spectrometry was used in conjunction with gel electrophoresis and liquid chromatography, to determine peptide sequences from American alligator (Alligator mississippiensis) leukocytes and to identify similar proteins based on homology. The goal of the study was to generate an initial database of proteins related to the alligator immune system. We have adopted a typical proteomics approach for this study. Proteins from leukocyte extracts were separated using two-dimensional gel electrophoresis and the major bands were excised, digested and analyzed by on-line nano-LC MS/MS to generate peptide sequences. The sequences generated were used to identify proteins and characterize their functions. The protein identity and characterization of the protein function were based on matching two or more peptides to the same protein by searching against the NCBI database using MASCOT and Basic Local Alignment Search Tool (BLAST). For those proteins with only one peptide matching, the phylum of the matched protein was considered. Forty-three proteins were identified that exhibit sequence similarities to proteins from other vertebrates. Proteins related to the cytoskeletal system were the most abundant proteins identified. These proteins are known to regulate cell mobility and phagocytosis. Several other peptides were matched to proteins that potentially have immune-related function.

2D separation of alligator leukocyte stained with ProteoSilver stain for mass spectrometry analysis. Approximately 400 ฮผg of leukocyte protein was loaded and separated on a 2D large gel format.
Primary structure of the 35 kDa lectin protein isolated from American alligator assembled from different endoprotease digestions. The peptide sequences were generated using ESIโ€“MS/MS. Peptides obtained from the different enzymes were highlighted using different colors.