Combined Enrichment/Enzymatic Approach To Study Tightly Clustered Multisite Phosphorylation on Ser-Rich Domains

The regulation of protein function through phosphorylation is often dominated by allosteric interactions and conformational changes. However, alternative mechanisms involving electrostatic interactions also regulate protein function. In particular, phosphorylation of clusters of Ser/Thr residues can affect protein-plasma membrane/chromatin interactions by electrostatic interactions between phosphosites and phospholipids or histones. Currently, only a few examples of such mechanisms are reported, primarily because of the difficulties of detecting highly phosphorylated proteins and peptides, due in part to the low ionization efficiency and fragmentation yield of multiphosphorylated peptides in mass spectrometry when using positive ion mode detection. This difficulty in detection has resulted in under-reporting of such modified regions, which can be thought of as phosphoproteomic dark matter. Here, we present a novel approach that enriches for multisite-phosphorylated peptides that until now remained inaccessible by conventional phosphoproteomics. Our technique enables the identification of multisite-phosphorylated regions on more than 300 proteins in both yeast and human cells and can be used to profile changes in multisite phosphorylation upon cell stimulation. We further characterize the role of multisite phosphorylation for Ste20 in the yeast mating pheromone response. Mutagenesis experiments confirmed that multisite phosphorylation of Ser/Thr-rich regions plays an important role in the regulation of Ste20 activity during mating pheromone signaling. The ability to detect protein multisite phosphorylation opens new avenues to explore phosphoproteomic dark matter and to study Ser-rich proteins that interact with binding partners through charge pairing mechanisms.