Deep Proteogenomics of a Photosynthetic Cyanobacterium

Cyanobacteria, the evolutionary ancestors of plant chloroplasts, contribute substantially to the Earth's biogeochemical cycles and are of great interest for a sustainable economy. Knowledge of protein expression is the key to understanding cyanobacterial metabolism; however, proteome studies in cyanobacteria are limited and cover only a fraction of the theoretical proteome. Here, we performed a comprehensive proteogenomic analysis of the model cyanobacterium Synechocystis sp. PCC 6803 to characterize the expressed (phospho)proteome, re-annotate known and discover novel open reading frames (ORFs). By mapping extensive shotgun mass spectrometry proteomics data onto a six-frame translation of the Synechocystis genome, we refined the genomic annotation of 64 ORFs, including eight completely novel ORFs. Our study presents the largest reported (phospho)proteome dataset for a unicellular cyanobacterium, covering the expression of about 80% of the theoretical proteome under various cultivation conditions, such as nitrogen or carbon limitation. We report 568 phosphorylated S/T/Y sites that are present on numerous regulatory proteins, including the transcriptional regulators cyAbrB1 and cyAbrB2. We also catalogue the proteins that have never been detected under laboratory conditions and found that a large portion of them is plasmid-encoded. This dataset will serve as a resource, providing dedicated information on growth condition-dependent protein expression and phosphorylation.