Systems Biology Analysis of Chlorella vulgaris Figure 1. Growth and lipid accumulation properties of C. vulgaris under nitrogen limitation. (A) Representative growth curves for C. vulgaris cultured photoautotrophically under nitrogen-replete (blue) and nitrogen-deplete (red) conditions. Harvest points utilized for all comparative analyses and images are circled. Inset: cultures grown under nitrogen-replete (green) and nitrogen-deplete (yellow) conditions. (B) Fatty acid methyl ester (FAME) analysis of C. vulgaris under nitrogen-replete and nitrogen-deplete conditions. (C) Average fatty acid composition (%, w/w) for C. vulgaris under nitrogen-replete and nitrogen-deplete conditions. (D) Epifluorescent microscopy images of BODIPY-stained C. vulgaris in nitrogen-replete (top panel) and nitrogen-deplete (bottom panel) medium. Red fluorescence is due to chlorophyll autofluorescence and green fluorescence is due to BODIPY-neutral lipid interaction. Magnification bar (white) equals 5 mm. doi:10.1371/journal.pone.0025851.g001 are the best possible BLAST results. The mean E-value was 3.22e- 06 and median E-value was 2e-38 (data not shown). Isolation and Fractionation of Soluble Proteome The cell wall of C. vulgaris contains an extensive network of biopolymers, largely composed of chitin and algaenan-like molecules, as well as uronic acid and proteoglycans (for extensive review of Chlorella sp. cell wall composition, refer to Takeda, 1991 [43]). The robust nature of this cell wall makes C. vulgaris extremely resistant to cell lysis, and in turn, hinders extraction of the soluble protein fraction. Numerous mechanical and chemical cell disruption methods were therefore examined in order to optimize cell lysis and increase sample complexity. Optimal lysis and protein extraction, determined by maximal lysate protein complexity and resolution on SDS-PAGE analysis, was ultimately obtained via multiple cycles of sonication. Increasing the number of sonication cycles improved protein identification rates by more than an order of magnitude (134 proteins from a single cycle, .2,000 proteins from six cycles). No cells remained intact when examined microscopically (data not shown). Beyond six cycles of sonication, protein abundance and complexity did not noticeably improve. Therefore, we utilized six cycles of sonication to maximize lysis efficiency and avoid potential protein degradation caused by excessive sonication treatment. The resulting SDS polyacrylamide gels demonstrate a high sample complexity, with a broad range of highly resolved proteins observed. Differential protein expression patterns between nitrogen-replete and nitro- gen-deplete conditions are clearly observed (Figure 3a). Proteomic Analysis and Annotation Proteomic analysis was performed using Gel-based liquid chromatography mass spectrometry (GeLC/MS, Figure 2). In- house Python and Awk scripts were used to annotate the de novo assembled C. vulgaris transcriptome using nucleotide blast (blastn) results and to properly format the resulting annotated transcrip- tome for use in Mascot. Each transcript isoform in the assembled transcriptome was annotated using the fasta header of the best blastn hit using Awk and Python codes (Figure 2). Since multiple PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e25851