Methods for and Insights from Phosphoproteome Analysis in Marine Microbes

Phosphorylation, the dynamic addition of a phosphate group to specific amino acids, is a key regulator of protein activity in both prokaryotes and eukaryotes. Protein phosphorylation is known to modulate nutrient acquisition, metabolism, growth and reproduction in model organisms, yet little is known about the role of phosphorylation marine organisms. Recent developments in LC-MS/MS make it possible to identify phosphorylation events in the proteome. We tested various methods in marine bacteria and developed a simple approach to phosphoproteome analysis. We then applied this method to cultured isolates of Prochlorococcus and diatom-associated Alteromonas sp. BB2AT2. We began by comparing the phosphoproteomes of these organisms in exponential and stationary phase growth. We conducted iterative experiments to assess completeness of our analysis, similar to the rarefaction approach used to determine sequence depth in ecology. We also explored semi-quantitative changes in protein phosphorylation when cells were subject to phosphate deplete media and/or phosphatase inhibitors. These early studies demonstrate the promise of phosphoproteomics to advance our understanding of bacterial biochemistry and microbe-environment interactions.