Dynamics of protein phosphorylation are regulated by the interplay of protein kinases and phosphatases. Current mass spectrometry-based phosphoproteomic approaches are extremely powerful in identifying and quantifying tens of thousands of different phosphorylation events in single biological samples. However, whereas the mapping of phosphorylation events has been successfully automated supporting high sample throughput, the characterization of responsible kinases and phosphatases still largely depends on laborious protein biochemical assays. To show direct (de)phosphorylation events in vitro kinase or phosphatase assays using single substrates or peptide arrays are still the methods of choice. Here we describe the development of an in vitro phosphatase assay using whole proteome under native conditions as input. We employ this approach to study the PP1 and PP2A target repertoire, characterizing thousands of potential target sites. Focusing on PPP2R5E/B56-containing complexes, we combine in vitro with in vivo phosphoproteomics to characterize bona fide target sites, which highlight PP2A`s role in regulating stress granule assembly.

This is an experiment linked to a publication about an on beads in vitro phosphatase assay to find direct phosphatase substrates. PRM was used to validate DIA results for 3 different proteins: eIF3A, eIF4B and DDX3X in A549 cells.

Samples from mammalian cell culture using A549 cells. No spike ins.