Wienkoop Stefanie, Weiss Julia, May Patrick, Kempa Stefan, Irgang Susann, Recuenco-Munoz Luis, Pietzke Matthias, Schwemmer Thorsten, Rupprecht Jens, Egelhofer Volker, Weckwerth Wolfram
Dept. of Molecular Systems Biology, University of Vienna, Vienna, Austria.
Mol Biosyst. 2010 Jun;6(6):1018-31. doi: 10.1039/b920913a. Epub 2010 Mar 31.
In the era of fast genome sequencing a critical goal is to develop genome-wide quantitative molecular approaches. Here, we present a metaproteogenomic strategy to integrate proteomics and metabolomics data for systems level analysis in the recently sequenced unicellular green algae Chlamydomonas reinhardtii. To achieve a representative proteome coverage we analysed different growth conditions with protein prefractionation and shotgun proteomics. For protein identification, different genome annotations as well as new gene model predictions with stringent peptide filter criteria were used. An overlapping proteome coverage of 25%, consistent for all databases, was determined. The data are stored in a public mass spectral reference database ProMEX (http://www.promexdb.org/home.shtml). A set of proteotypic peptides comprising Calvin cycle, photosynthetic apparatus, starch synthesis, glycolysis, TCA cycle, carbon concentrating mechanisms (CCM) and other pathways was selected from this database for targeted proteomics (Mass Western). Rapid subcellular fractionation in combination with targeted proteomics allowed for measuring subcellular protein concentrations in attomole per 1000 cells. From the same samples metabolite concentrations and metabolic fluxes by stable isotope incorporation were analyzed. Differences were found in the growth-dependent crosstalk of chloroplastidic and mitochondrial metabolism. A Mass Western survey of all detectable carbonic anhydrases partially involved in carbon-concentrating mechanism (CCM) revealed highest internal cell concentrations for a specific low-CO2-inducible mitochondrial CAH isoform. This indicates its role as one of the strongest CO2-responsive proteins in the crosstalk of air-adapted mixotrophic chloroplast and mitochondrial metabolism in Chlamydomonas reinhardtii.
在快速基因组测序时代，一个关键目标是开发全基因组定量分子方法。在此，我们提出一种元蛋白质组学策略，用于整合蛋白质组学和代谢组学数据，以便对最近测序的单细胞绿藻莱茵衣藻进行系统水平分析。为实现具有代表性的蛋白质组覆盖范围，我们通过蛋白质预分级和鸟枪法蛋白质组学分析了不同的生长条件。在蛋白质鉴定方面，使用了不同的基因组注释以及具有严格肽段筛选标准的新基因模型预测。确定了所有数据库一致的25%的重叠蛋白质组覆盖范围。数据存储在公共质谱参考数据库ProMEX（http://www.promexdb.org/home.shtml）中。从该数据库中选择了一组包含卡尔文循环、光合装置、淀粉合成、糖酵解、三羧酸循环、碳浓缩机制（CCM）和其他途径的蛋白质型肽段用于靶向蛋白质组学（质谱免疫印迹法）。快速亚细胞分级分离与靶向蛋白质组学相结合，能够测量每1000个细胞中阿托摩尔水平的亚细胞蛋白质浓度。对相同样本通过稳定同位素掺入分析了代谢物浓度和代谢通量。发现叶绿体和线粒体代谢的生长依赖性串扰存在差异。对部分参与碳浓缩机制（CCM）的所有可检测碳酸酐酶进行的质谱免疫印迹调查显示，一种特定的低二氧化碳诱导型线粒体CAH同工型在细胞内浓度最高。这表明它在莱茵衣藻适应空气的混合营养叶绿体和线粒体代谢串扰中作为最强的二氧化碳响应蛋白之一发挥作用。
