RESEARCH ARTICLE Multidimensional analysis of the insoluble sub-proteome of Oceanobacillus iheyensis HTE831, an alkaliphilic and halotolerant deep-sea bacterium isolated from the Iheya ridge Robert L. J. Graham 1 , Catherine E. Pollock 1 , S. Naomi O’Loughlin 1 , Nigel G. Ternan 1 , D. Brent Weatherly 2 , Rick L. Tarleton 2 and Geoff McMullan 1 1 School of Biomedical Sciences, University of Ulster, Coleraine, County Londonderry, UK 2 The Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA We report the first proteomic analysis of the insoluble sub-proteome of the alkaliphilic and halotolerant deep-sea bacterium Oceanobacillus iheyensis HTE831. A multidimensional gel-based and gel-free analysis was utilised and a total of 4352 peptides were initially identified by auto- mated MS/MS identification software. Automated curation of this list using PROVALTreduced our peptide list to 467 uniquely identified peptides that resulted in the positive identification of 153 proteins. These identified proteins were functionally classified and physiochemically char- acterised. Of 26 proteins identified as hypothetical conserved, we have assigned function to all but four. A total of 41 proteins were predicted to possess signal peptides. In silico investigation of these proteins allowed us to identify three of the five bacterial classes of signal peptide, namely: (i) twin-arginine translocation; (ii) Sec-type and (iii) lipoprotein transport. Our proteomic strategy has also allowed us to identify, at neutral pH, a number of proteins described previously as belonging to two putative transport systems believed to be of importance in the alkaliphilic adaptation of O. iheyensis HTE831. Received: September 1, 2006 Accepted: October 4, 2006 Keywords: Bacteria / ESI-MS/MS / LC-MS/MS 82 Proteomics 2007, 7, 82–91 1 Introduction In 1818, Sir John Ross made the first successful attempt to ascertain the limit of life at depths greater than five or six hundred feet when he brought up several pounds of a greenish mud from a depth of 1920 m in Baffin’s Bay, Canada; however, no accurate determination of the nature of this mud could be made [1]. It was almost two centuries later that the submersible Kaiko touched down at the bottom of the Challenger Deep in the Mariana Trench at a depth of 10 897 m and scooped up mud from this deepest seafloor environment. Investigations of this mud found it to be ‘home’ to numerous microorganisms, including pyschro- philes, thermophiles, halophiles and also non-extremophilic microbes [2]. Oceanobacillus iheyensis HTE831 was originally isolated from deep-sea sediment collected at a depth of 1050 m on the Iheya Ridge off the Nansei Islands [3]. It is a member of the novel genus Oceanobacillus, literally meaning ‘the ocean bacillus/rod’. O. iheyensis HTE831 is the first Gram-positive spore-forming isolate from the bathypelagic zone and exhib- its extremely halotolerant (0–21% NaCl), facultatively alkali- philic (pH 6.5–10) and piezotolerant (0–30 MPa) phenotypes. Correspondence: Dr. Robert Graham, School of Biomedical Science, University of Ulster, Cromore Road, Coleraine, BT52 1SA, UK E-mail: rl.graham@ulster.ac.uk Fax: 144-2870-324375 Abbreviation: FDR, false-discovery rate DOI 10.1002/pmic.200600665  2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com