EVALUATION OF CAROTENOIDS' IMPACT ON PROTEIN EXPRESSION IN CYANOBACTERIA BY LABEL-FREE PROTEOMICS

Purpose: Identification of carotenoid-regulated proteins in cyanobacteria by mass spectrometry-based proteomics. Methods: Strains were derived from Synechocystis sp. PCC 6803 (wild type, WT). WT and ΔcrtH/B mutant cells were cultivated at 30℃ in light-activated heterotrophic conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on crude cellular protein extracts, and gels were cut horizontally into pieces for subsequent in-gel tryptic digestion and reversed-phase liquid chromatography (LC) coupled to data-dependent electrospray ionization (ESI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) using nanoLC (Eksigent) and LTQFT (Thermo) instruments. MS/MS spectra were extracted by BioWorks and searched against a composite protein sequence database of cyanobacteria using Mascot. Quantitative protein expressions were obtained through MS/MS spectral counts determined through Scaffold and by extracted peptide intensity features generated with the Progenesis LC-MS software. Results: Using the Synechocystis sp. PCC 6803 ΔcrtH/B mutant, our "GeLC-MS/MS" analyses permitted global-scale identification of protein expression changes in carotenoid-regulated cells by label-free approaches. Overall, 497 proteins were identified in this genetically modified strain of cyanobacteria. Considering at least a 2-fold change using the total number of MS/MS spectra identified for a particular protein (Scaffold), 60 carotenoid-regulated proteins were obtained from G-tests (p<0.05). As an alternative data-processing method, Progenesis LC-MS was used to process the same set of raw data files by alignment and normalization of MS peak intensities, data filtering for charge states and, then, exporting peak lists to query against the cyanobacteria protein database using Mascot. When requiring at least a 2-fold change in expression and excluding hypothetical hits, this latter approach afforded 197 carotenoid-regulated proteins by analysis of variance (ANOVA, p<0.05). From genome information on Synechocystis (a part of CyanoBase, an online resource for cyanobacterial genomes), these proteins were found to be involved in photosynthesis and respiration, transport and binding, transcription and translation, and biosynthesis. Conclusions: Altogether, our GeLC-MS/MS analyses permitted a successful quantitative survey of carotenoids' global impact on differential expressions of key cellular proteins in the ΔcrtH/B mutant versus WT Synechocystis sp. PCC 6803 for the first time.