William J. Simon 1 John J. Hall 1 Iwane Suzuki 2 Norio Murata 2 Antoni R. Slabas 1 1 School of Biological and Biomedical Sciences, University of Durham, Durham, UK 2 Department of Regulation Biology, National Institute of Basic Biology, Okazaki, Japan Proteomic study of the soluble proteins from the unicellular cyanobacterium Synechocystis sp. PCC6803 using automated matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting The unicellular cyanobacteria Synechocystis sp. (PCC6803) has become a model organism for a range of biochemical and molecular biology studies aimed at investigat- ing environmental stress responses. In this study the soluble proteins of Synechocystis were analysed using narrow pH range (pH 4.5–5.5) zoom gels, automated matrix- assisted laser desorption/ionization mass spectrometry acquisition, spectral proces- sing and database searching. The work sets the foundation for investigations of pro- teomic changes following stress treatment. One hundred and ninety-two protein spots were analysed and 105 proteins identified, of these 37 were novel proteins not pre- viously seen on two-dimensional gels. Proteins involved in amino acid biosynthesis, energy metabolism and protein modification were identified using this fully automated procedure demonstrating that automated acquisition and processing will be a useful tool for proteomic analyses on this organism. Keywords: Automated matrix-assisted laser desorption/ionization-mass spectrometry analysis / Cyanobacteria / Synechocystis PRO 0309 1 Introduction Cyanobacteria (blue green algae) are a major diverse group of organisms of scientific importance because they are photosynthetic prokaryotes and are believed to be the ancestral origin of the higher plant chloroplast [1]. The phyla ranges from single unicellular genera such as Synechocystis through to complex multicellular filamen- tous strains, many of which have specialised differen- tiated cells, such as the heterocyst in nitrogen fixing Ana- baena and Nostoc. They are characterised by their ability to tolerate extreme environments, such as desiccation, extreme pH, high temperatures and high salinity levels. The unicellular strain Synechocystis (PCC 6803) has become a model organism for a range of biochemical and molecular biology studies that include work on photosynthesis [2], osmotic stress responses [3–5], and chilling and heat shock responses [6, 7]. The completion of the nucleotide sequencing of the entire genome of this organism [8] has led to the construction of a database (CyanoBase) of 3168 predicted genes [9]. Analyses of these genes using GeneQuiz [10] assigned a function to 64% of these proteins by homology to proteins in data- bases from other organisms, confirmation of function will require further evidence, 18% have homology to proteins of no known function and 18% have no known homo- logues. One-third of all of the proteins in Synechocystis are believed to be membrane associated. Aqueous poly- mer two phase partitioning has been used to isolate pure plasma and thylakoid membranes as a prelude to a pro- teomic study of the membrane component of Synecho- cystis [11]. DNA microarrays containing 95% of the genes of Syne- chocystis sp. (PCC6803) are available and these have been used to identify stress responsive gene expression during acclimation from low to high light [12] and in response to cold [13] and salt stress [14]. In the light accli- mation experiments, more than 160 responsive genes were identified which included up- and down-regulated genes involved in the photosynthetic machinery (psa, psb, apc and cpc) and the up-regulation of genes in- volved in CO 2 fixation (rbc, ccm and ndh). Cold inducible responsive genes identified included the genes for acyl lipid n12 (desA), n6 (desD) and o3 (desB) desaturases, responsible for enhanced membrane fluidity, histidine kinases, Hik 33 and Hik 19 involved in the perception and signal transduction of decreases in temperature, and genes for components of the translational and transcrip- tional machinery. In comparison to almost entire genome Correspondence: Professor A. R. Slabas, School of Biological and Biomedical Sciences, University of Durham, Science Laboratories, South Road, Durham DH1 3LE UK E-mail: a.r.slabas@durham.ac.uk Fax: +44-191-3747395 Abbreviation: PS1, proteomic solutions 1 software Proteomics 2002, 2, 1735–1742 1735  2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/02/1212–1735 $17.501.50/0