Systems Biology Analysis of Chlorella vulgaris Figure 6. Improved pathway identification using a de novo assembled transcriptome database and changes in protein abundance under nitrogen depletion. (A) Critical components of the fatty acid and triacylglycerol (TAG) biosynthetic pathways were absent from initial MS/ MS searches against all available Chlorophyta databases. Proteins highlighted in yellow were amongst the proteins absent from initial analyses, yet positively identified when searching against the C. vulgaris transcriptome database. Proteins in green (not highlighted) were identified using both Chlorophyta and C. vulgaris transcriptome databases. Numbers below proteins represent NSAF values (105) for nitrogen replete and nitrogen deplete conditions, respectively. ACCase, acetyl-CoA carboxylase; ACP, acyl carrier protein; AMPK, AMP-activated kinase; DAGK, diacylglycerol kinase; DGAT, diacylglycerol acyltransferase; DHAP, dihydroxyacetone phosphate; ENR, enoyl-ACP reductase; FATP, fatty acyl-ACP thioesterase (putative); G3PDH, glycerol-3-phosphate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase; HD, 3-hydroxyacyl-ACP dehydratase; KAR, 3-ketoacyl-ACP reductase; KAS, 3-ketoacyl-ACP synthase; LPAAT, lyso-phosphatidic acid acyltransferase; LPAT, lyso-phosphatidylcholine acyltransferase; MAT, malonyl-CoA:ACP transacylase; PAP, phosphatidic acid phosphatase. Adapted from Radakovits et al., 2010 and Hu et al., 2008. (B) Corresponding spectral count fold-changes for components of the FA (left panel) and TAG (right panel) biosynthetic components. doi:10.1371/journal.pone.0025851.g006 for biodiesel or renewable diesel and jet fuel. Our experimental conditions also yield ,60% fatty acids on a dry cell-weight basis, nearly 3-fold higher than wild type C. reinhardtii, suggesting it is an excellent model system to examine changes in gene and protein expression under conditions that induce high-oil accumulation. We and our co-workers have had limited success reproducing the transformation techniques with C. vulgaris reported by Jarvis and Brown [31] and Chow and Tung [30], but we have recently improved on these methods and have developed a simple and reproducible transformation protocol (J.J. Lee and Y.-C. Chou, unpublished results). The concurrent development of systems biology and genetic tools will help establish C. vulgaris as a viable model organism for algal biofuels development. The utility of a de novo assembled transcriptome as a proteomic search model Proteomic analysis using orthologous sequence databases presents a unique challenge in that it requires nearly identical m/z values (61–2 Da) between the search model and peptides of interest in order to positively match an equivalent m/z ratio of statistical significance. As such, a single amino acid differential between a search model sequence and a peptide fragment sequence of interest can often result in a failure to produce a statistically significant match, leaving significant gaps in protein identification. One example of an absence caused by a single amino acid differential was observed for ACAT. Using Chlorophyta sequence databases, ACAT was not identified via proteomic analysis, yet was successfully identified using the C. vulgaris PLoS ONE | www.plosone.org 10 October 2011 | Volume 6 | Issue 10 | e25851