Phosphoproteomics

While phosphoproteomics will greatly expand knowledge about the numbers and types of phosphoproteins, its greatest promise is the rapid analysis of entire phosphorylation based signalling networks.

[3] A sample large-scale phosphoproteomic analysis includes cultured cells undergo SILAC encoding; cells are stimulated with factor of interest (e.g. growth factor, hormone); stimulation can occur for various lengths of time for temporal analysis, cells are lysed and enzymatically digested, peptides are separated using ion exchange chromatography; phosphopeptides are enriched using phosphospecific antibodies, immobilized metal affinity chromatography or titanium dioxide (TiO2) chromatography; phosphopeptides are analyzed using mass spectrometry, and peptides are sequenced and analyzed.

This is due to the optimization of enrichment protocols for phosphoproteins and phosphopeptides, better fractionation techniques using chromatography, and improvement of methods to selectively visualize phosphorylated residues using mass spectrometry.

[5] Previous procedures to isolate phosphorylated proteins included radioactive labeling with 32P-labeled ATP followed by SDS polyacrylamide gel electrophoresis or thin layer chromatography.

The two main procedures to perform this task are using isotope-coded affinity tags (ICAT) and stable isotopic amino acids in cell culture (SILAC).

The ability to measure the global phosphorylation state of many proteins at various time points makes this approach much more powerful than traditional biochemical methods for analyzing signalling network behavior.

The researchers were able to use this information in combination with previously published data to construct a signal transduction network for the proteins downstream of the EphB2 receptor.

Another recent phosphoproteomic study included large-scale identification and quantification of phosphorylation events triggered by the anti-diuretic hormone vasopressin in kidney collecting duct.

Increasing amounts of data are available suggesting that distinctive phosphoproteins exist in various tumors and that phosphorylation profiling could be used to fingerprint cancers from different origins.

In addition, systematic cataloguing of tumor-specific phosphoproteins in individual patients could reveal multiple causative players during cancer formation.