Proteomic analysis of the neutrophil proteins of the tammar wallaby (Macropus eugenii)

A proteomic analysis of neutrophils from the tammar wallaby, Macropus eugenii, has been performed. Neutrophils were isolated from peripheral blood using density gradient centrifugation with Histopaque-1077, followed by treatment with ammonium chloride to lyse residual erythrocytes. Two-dimensional gel electrophoresis (2-DE) of lysed neutrophils was undertaken followed by in-gel trypsin digest and nanoliquid chromatography coupled tandem mass spectrometry (LC-MS) analysis and database searches. Seventy-seven proteins were isolated, 53 of which could be identified with high confidence as primarily of cytosolic origin. Protein identifications were only possible by matching identical peptide sequences within the NCBInr mammalian database with the Mascot search program. Sequence identities were only deemed acceptable if more than three peptides were identified, the precursor/protein ion tolerances were less than ± 0.25 Da and the total Mowse scores were greater than 100. The validity of this approach was tested using a scrambled database where no single identified peptide showed Mowse scores greater than 55. This is the first report of the neutrophil proteins of any marsupial and represents a first step in examining the identity of proteins involved in innate defence in this marsupial.