Simplified basic protocol for phosphoproteomic analysis. A protein sample is subjected to proteolysis with a purified, recombinant protease (typically trypsin). Phosphopeptides are enriched through use of ion chromatography, e.g., with TiO₂ or Fe-IMAC columns, which select peptides with negative charges. The column eluate is subjected to LC-MS/MS analysis to identify and quantify phosphopeptides, often after additional fractionation (not shown). IMAC, immobilized metal affinity chromatography. Circled P indicates phosphorylation.

Mapping of vasopressin-responsive phosphoproteins to major physiologic effects of vasopressin at a cellular level. Proteins are indicated by their official gene symbols. Those showing increases in phosphorylation in response to vasopressin are indicated in green, whereas those showing decreases are indicated in red. Known PKA targets are underlined.

PKA signaling network. PKA target phosphoproteins were identified by deletion of PKA by genome editing techniques followed by SILAC-based phosphoproteomics (Isobe et al., 2017). Functional groups around the periphery correspond to vasopressin-regulated processes in Table 1. Yellow nodes indicate other protein kinases that undergo PKA-mediated phosphorylation. Gray nodes indicate non-PKA targets included to consolidate phosphoprotein clustering. Analysis used a computer application called STRING (https://string-db.org/) for initial mapping followed by additional classification by Gene Ontology biologic process terms. Final visualization used Cytoscape (https://cytoscape.org/).

Lithium signaling network. Phosphoproteins altered in rat inner medullary collecting ducts by in vivo lithium administration were clustered according to protein function. Red nodes indicate proteins with phosphosites downregulated by lithium; green nodes indicate proteins with phosphosites that were upregulated by lithium. Nodes with thick borders are protein kinases. Original data were from Trepiccione et al. (2014).