Proteomic analysis of the neutrophil proteins of the tammar wallaby (Macropus eugenii) Kiran Ambatipudi a , Julie Old a , Michael Guilhaus b , Mark Raftery b , Lyn Hinds c , Elizabeth Deane a, ⁎ a Department of Biological Sciences, Division of Environmental and Life Sciences, Macquarie University, NSW, 2109, Australia b Bioanalytical Mass Spectrometry Facility, University of NSW, NSW 2052. Australia c CSIRO Division of Entomology, Canberra, GPO Box 1700, Canberra, ACT 2601, Australia Received 15 January 2006; received in revised form 27 April 2006; accepted 2 May 2006 Available online 3 June 2006 Abstract 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. © 2006 Elsevier Inc. All rights reserved. Keywords: Marsupials; Neutrophils; Electrospray ionisation-tandem mass spectrometry; Two-dimensional gel electrophoresis 1. Introduction Polymorphonuclear neutrophils are short-lived phagocytic cells that play a pivotal role in innate defence in eutherian mammals. Along with the other major phagocytic cell type, the macrophage, these cells are of ancient evolutionary origin, arising from the wandering phagocytic amoebocytes of inver- tebrates (Roitt et al., 2001). In eutherian mammals, neutrophils kill invading microorganisms by two mechanisms. The first method consists of an oxidative burst mediated via NADPH oxidase, which results in a cascade of reactive species including superoxide anions, subsequent production of hydrogen per- oxide, release of myeloperoxidase (MPO) and the generation of hypochlorous acid, all of which result in oxidative damage to microorganisms (Avram et al., 2004). The second mode of action depends on the controlled release of a repertoire of gene-coded antimicrobial polypeptides contained within neutrophil gran- ules. These peptides kill by interaction with the phospholipid bilayers of the cell membranes of the ingested microorganisms, resulting in membrane permeabilisation, loss of ions and metabolites, and blockade of various essential cellular functions (Blondelle et al., 1999; Hancock and Chapple, 1999). The advent of proteomic techniques has facilitated the study of protein expression in a range of cell types. However, to date, neutrophil proteins have only been documented in humans (Boussac and Garin, 2000; Fessler et al., 2002; Lominadze et al., 2005), the rat (Piubelli et al., 2002) and most recently in the cow (Lippolis and Reinhardt, 2005). Of these the most com- prehensive analysis has been undertaken on human neutrophils with over 500 proteins identified from subcellular organelles (Lominadze et al., 2005). In the latter species, this extensive Comparative Biochemistry and Physiology, Part D 1 (2006) 283 – 291 www.elsevier.com/locate/cbpd Abbreviations: MPO, myeloperoxidase; HSP, heat shock protein; fMLP, N-formyl-methionyl-leucyl-phenylalanine. ⁎ Corresponding author. Tel.: +61 2 9850 8418; fax: +61 2 9850 9671. E-mail address: edeane@els.mq.edu.au (E. Deane). 1744-117X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbd.2006.05.002