Proteome-Wide Analysis and Diel Proteomic Profiling of the Cyanobacterium Arthrospira platensis PCC 8005 Sabine Matallana-Surget 1 , Je ´re ´ my Derock 1 , Baptiste Leroy 1 , Hane ` ne Badri 1,2 , Fre ´de ´ ric Deschoenmaeker 1 , Ruddy Wattiez 1 * 1 Department of Proteomics and Microbiology, Interdisciplinary Mass Spectrometry Center (CISMa), University of Mons, Mons, Belgium, 2 Unit of Microbiology, Expert Group Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK CEN), Mol, Belgium Abstract The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation. Citation: Matallana-Surget S, Derock J, Leroy B, Badri H, Deschoenmaeker F, et al. (2014) Proteome-Wide Analysis and Diel Proteomic Profiling of the Cyanobacterium Arthrospira platensis PCC 8005. PLoS ONE 9(6): e99076. doi:10.1371/journal.pone.0099076 Editor: Joshua L. Heazlewood, Lawrence Berkeley National Laboratory, United States of America Received December 17, 2013; Accepted May 9, 2014; Published June 10, 2014 Copyright: ß 2014 Matallana-Surget et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the European Space Agency (ESA-PRODEX) and the Belgian Federal Office. This work was also partially financed by the FNRS under grant ‘‘grand equipement’’ no. 2877824. Fre ´de ´ric Deschoenmaeker is an F.R.I.A. PhD student. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ruddy.wattiez@umh.ac.be Introduction Arthrospira platensis, better known as ‘‘spirulina’’, is a motile non- heterocystous, non-N(2)-fixing cyanobacterium, that forms of a multicellular coiled filament, and it is typically found in alkaline lakes [1]. Arthrospira species provide exceptional nutritional value with high protein content (50–70% of its dry weight), it is rich in essential fatty acids, produces a variety of minerals, vitamins, and nutritional pigments such as phycocyanin [2] and thus has been proposed as a potential tool to manage the problem of malnutrition in developing countries. The A. platensis PCC 8005 strain was selected by the European Space Agency (ESA) for long- term space missions as a primary oxygen producer and also as an accessory balanced food provider for human crew survival in its Micro-Ecological Life Support System Alternative (MELiSSA) [3]. Therefore, proteomic studies of MELiSSA organisms appeared essential to complete the global behavior profile of these microorganisms in certain culture conditions. The analysis of cyanobacterial proteins has been traditionally conducted using the proteome, primarily employing electropho- resis-based approaches [4–12]. Shotgun proteomics analysis involves the use of multidimensional protein/peptide separation to fractionate complex protein/peptide mixtures, thus simplifying the peptide samples for LC-MS/MS and enabling acquisition of MS/MS spectra for lower abundance peptides. The value of the use of such multi-faceted workflows was first demonstrated on the proteome of Synechocystis sp. PCC 6803 by Gan and co-workers [13]. Here, we report a shotgun proteomics study of A. platensis using a combination of LC-MS/MS approaches with gel-free and gel-based protein/peptide fractionation steps, such as one-dimen- sional gel electrophoresis (SDS-PAGE and IEF), 2D and 3D LC- MS/MS (Cu-IMAC) of the soluble and/or membrane and secreted protein fractions. Whole-genome sequencing of strain PCC 8005 and its annotation has been recently completed, and thus provide key assets to facilitate proteomics approaches [14]. Our present work adds new perspectives in the field of shotgun proteomics by developing a new fractionation method (Cu-IMAC) allowing the depletion of the most abundant proteins (phycocy- anins) and enabling the detection of low abundance proteins. Another key point to increase the proteome coverage of A. platensis consists of simply diversifying experimental growth conditions, such as cultivating the cells under different light conditions, i.e. shifting from continuous light to a 12-hour light/dark (LD) cycle. This latter observation remains obvious but is still, unfortunately, too often ignored. This could represent a ‘biological fractionation’ performed by the organism itself, with all the proteins not being constitutively expressed or being exposed to the same conditions [15]. PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e99076