Targeted proteome analysis of microalgae under high-light conditions by optimized protein extraction of photosynthetic organisms

Abstract

Cell disruption and protein solubilization protocols for the relative quantification of individual subunits in photosystems were developed for photosynthetic organisms including cyanobacterium Synechocystis sp. PCC 6803, green-algae Chlamydomonas reinhardtii, and seed plant Arabidopsis thaliana. The optimal methods for the disruption of Chlamydomonas, Synechocystis, and Arabidopsis cells were sonication, microbeads (Φ approximately 0.1mm), and large beads (Φ=5.0mm), respectively. Extraction of the total proteins exceeded 90% using each optimal cell disruption method. Solubilization efficiency of membrane proteins was improved by the phase transfer surfactant (PTS) method. Ninety seven and 114 proteins from Chlamydomonas and Synechocystis, respectively, including membrane proteins such as photosystem proteins, ATP synthase, and NADH dehydrogenase, were successfully analyzed by nano-liquid chromatography tandem mass spectrometry. These results also indicated the improved efficiency of solubilization and trypsin digestion using PTS buffer. The results of the relative quantitative evaluation of photosystem subunits in Chlamydomonas and Synechocystis grown under high-light conditions were consistent with those of previous studies. Thus, the optimal cell disruption and PTS methods allow for comprehensive relative quantitative proteome analysis of photosynthetic organisms. Additionally, NdhD1 and NdhF1, which are NDH-1 subunit homologs, were increased under high-light conditions, suggesting that the NDH-1L complex, including NdhD1 and NdhF1, is increased under high-light conditions. The relative quantitative proteome analysis of individual subunits indicates the diverse functions of NDH-1 protein.