Overview

1. Day/night cycle analysis and first protein map of the cyanobacteriumArthrospiraPCC8005 assessed by extensive shotgun and quantitative proteomics approaches Sabine Matallana-Surget, Baptiste Leroy, Jerémy Derock, Ruddy Wattiez* Department of Proteomic and Microbiology, University of Mons,, Mons, Belgium Overview This study assesses progress in cyanobacterialshotgun proteomics, and focuses specially on the proteome of the cyanobacteriumArthrospira PCC8005, a strain of particular interest for the European Space Agency (ESA). This work adds new perspectives in the field of shotgun proteomics by developing a set of methods allowing to get rid of the most abundant proteins (phycocyanin, phycobiliproteins) and access under-represented proteins of a complex sample.  Introduction Methods Arthrospiraplatensis, better known as “spirulina”, is a motile cyanobacterium, made up of a multicellular coiled filament, found in the ground as well as in water-salt, brackish or lake water. Owing to its exceptional nutritional value, A. platensiswas used to face the problem of denutrition in developing countires. It is today used by astronauts on their long-term missions in orbit and is thus a strain of particular interest for the European Space Agency in the MELiSSA project. A. platensisis studied in Mons as a part of the European MEliSSA (Micro-Ecological Life Support System Alternative) project, which aims at developing a closed artificial ecosystem, intended to be used aboard long term manned missions to Mars, in order to recycle wastes and to produce a balanced food. This study presents the first protein of this key cyanobacterium by using a combination of different methods on the soluble and/or membrane secreted proteome. We combined LC-MS/MS approaches with gel-free and gel-based protein/peptides fractionation steps: one-dimensional gel electrophoresis (SDS PAGE and IEF), 2D and 3D-LC MS/MS (IMAC-Copper) on the soluble and/or membrane and secreted fractions.  In order to increase the proteome coverage, we also cultivated this cyanobacterium under different light conditions, shifting from continuous light to a 12-hour light/dark cycle and quantitative proteomics was assessed using an ICPL post-digest labeling ShotgunProteomics 3D-LC-MS/MS ShotgunProteomics - Comparison Flowthrough fraction (218) Elution fraction (676) A total of 1307 proteins was identified, representing 22% coverage of the theoretical proteome. In addition, 450 identified proteins which were previously either unknown or considered to be hypothetical were shown to be true gene products in Arthrospira PCC8005. The identified proteins participate in the major metabolic and cellular processes of cyanobacterial cells during the exponential growth phase, under continuous light exposure. 109 109 567 A novel aspect of this study is that a 3D LC-MS/MS was used to reduce the complexity of our protein extract and also allowed the examination of the binding behavior to copper of several proteins. We observed a significant difference comparing both flowthrough and elution fractions, with two major bands in the flowhrough, corresponding to phycocyanin proteins. A proteinmap of all theoreticalproteins in Arthrospira PCC8005 and expressedproteinsidentified in all the experiments. 250 150 100 75 50 37 25 20 15 10 Quantitative proteomics Percentage of proteinsgrouped by Clusters of Orthologous Groups (COGs) * When the 4 approaches cited above were compared in term of non-redundant identified proteins, a relative high overlap between the techniques was observed. However, a significant portion of specific proteins for both 2D and 3DLC-MS/MS with immobilized metal affinity chromatography (IMAC-Copper) was observed, with 266 and 123 specific proteins respectively. * * Venn diagram showing non-redundant proteins identified in the 2D-LC, 3D-LC the IPG-IEF-LC and the SDS-PAGE-LC MS/MS approaches. IEF (560) * 311 proteinswerefound to beaffected by a light-dark cycle. 45 33 1 32 154 261 4 30 143 55 15 123 266 86 56 The ratios of differential expression are rangingfrom 0.2 (ferritin-likeprotein) to 4.2 (Glycine-rich RNA-bindingprotein). The study of the light-dark cycle, assessedusing an ICPL post-digest method, added 270 newlyidentifiedproteins to the total number of proteinscharacterized by 2D-LC-MS/MS. IMAC-Cu2+ (785) Proteinsfrom the phycobilisome (red) (light harvestingantennae) were, as expected, in higherabundance in light condition. However, proteinsfrom the 2 photosystems (blue) were all in higherabundance in the dark condition. This couldreflect a increased turnover of thisproteinshighlychallenged in light condition. 2D-LC (1043) SDS (511) Conclusion This study has highlighted new characteristics of Arthrospiraplatensisproteome. The identified expressed proteome was higher than the percentage of proteome coverage found in other cyanobacterial proteome studies. Proteins mostly affected during a light-dark cycle belonged to the translational engineering pathway, energy production and carbohydrate transport/metabolism. The obtained results gave insights into the functional and metabolic processes occurring in an important model organism, and may additionally be significant for the manual annotation of the proteome of Arthrospira PCC8005. Aknowledgments Fundings for thisprojectwasprovided by an ESA Grant .