A sneak preview of the daily life of S. elongatus by MS-based proteomics

Abstract

Circadian rhythms are self-sustained and adjustable cycles, typically entrained with light/dark and/or temperature cycles. These rhythms are present in animals, plants, fungi and several bacteria. The central mechanism behind these ‘pacemakers’ and the connection to the circadian regulated pathways are still poorly understood. The circadian rhythm of the cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) is highly robust and controlled by only three proteins, named KaiA, KaiB and KaiC. This central clock system has been extensively studied functionally, structurally and can be reconstituted in vitro. These characteristics together with the relatively small genome (2.7 Mbp) of S. elongatus, make it an ideal model system for the study of circadian rhythms. To provide new insights for the molecular mechanisms of the S. elongatus circadian clock in particular, different quantitative proteomics and phosphoproteomics methods were used. First, high-resolution Mass Spectrometry (MS) was combined with the TMT 6-plex labeling technique to probe cyclic protein abundance variations across the S. elongatus proteome. This 48-hour time-series study revealed that, in contrast to abundant occurrences of cyclic and even circadian rhythms at the transcript level (30-60%), at the protein level these variations are much less pronounced, with only 5% of the quantified proteome showing significant cyclic variations. Next, the focus moved towards the analysis of protein interactions and complexes. A novel approach, combining native size exclusion chromatography and MS, was used to uncover differences in protein assemblies between light and dark states. Several proteins involved in higher order complexes, which didn’t show diurnal adaptation in protein abundance in the previous study, were found to have association dynamics. Some examples are the photosynthetic and ribosomal proteins, which showed differences in abundances as well as variable assemblies. Finally, the combination of MS, Ti4+-IMAC phosphopeptide enrichment and TMT 6-plex labeling was explored to uncover clues for regulation of molecular rhythms by protein phosphorylation. This first look at the phosphoproteome revealed new phosphorylation sites on proteins associated with diverse molecular pathways.