Two approaches have been used to isolate and identify proteins of the granules of neutrophils of the tammar wallaby, Macropus eugenii. Stimulation with PMA, Ionomycin and calcium resulted in exocytosis of neutrophil granules as demonstrated with electron microscopy. However proteomic analysis using two dimensional gel electrophoresis, in-gel trypsin digestion followed by nano liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) failed to identify any anticipated granule proteins in the reaction supernatants. Subsequent use of differential centrifugation and lysis followed by the application of the same proteomic analysis approach resulted in the isolation and confident identification of 39 proteins, many of which are known to be present in the granules of neutrophils of eutherian mammals or play a role in degranulation. These proteins notably consisted of the known antimicrobials, myeloperoxidase (MPO), serine proteinase, dermcidin, lysozyme and alkaline phosphatase. A number of important known antimicrobials, however, were not detected and these include defensins and cathelicidins. This is the first report of the neutrophil granule proteins of any marsupial and complements previous reports on the cytosolic proteins.
- Electrospray ionisation-tandem mass spectrometry
- Two-dimensional gel electrophoresis