Deep proteogenomics of a unicellular cyanobacterium

Cyanobacteria are photoautotrophs that profoundly impact the biogeochemical cycles on Earth. Due to their photosynthetic lifestyle that includes the fixation of atmospheric CO2, they are of increasing interest for a sustainable economy. Knowledge of protein expression and regulation is key for understanding of the cyanobacterial metabolism; however, proteome studies in cyanobacteria are still limited and cover only a fraction of the theoretical proteome. Here, we performed a proteogenomic analysis of 628 LC-MS/MS measurements for the unicellular model cyanobacterium Synechocystis sp. PCC 6803 to characterize the expressed (phospho)proteome, re-annotate known and discover potential novel open reading frames (ORFs). By mapping extensive shotgun MS proteomics data generated by the SCyCode consortium onto a six-frame translation of the Synechocystis genome, we re-annotated 96 start sites and discovered 103 novel open reading frames (ORFs). Through re-analysis of previously published multi-omics datasets, we confirmed 48 re-annotated or novel ORFs with high confidence. Our study resulted in the largest reported proteome and phosphoproteome dataset for Synechocystis, covering expression of about 80% of the theoretical proteome and 642 O-phosphorylation events under various cultivation conditions, such as nitrogen or carbon limitation. This dataset will serve as a resource providing dedicated information on condition-dependent protein expression and phosphorylation.