Pan, L. et al. J. Am. Chem. Soc. 134, 18201–18204 (2012).

Phosphorylation-site stoichiometry is important for understanding the functional implications of phosphorylation dynamics in the cell. However, an apparent increase in phosphorylation, for example, could simply reflect an increase in concentration of the protein of interest under a changing biological condition. It is therefore important to normalize for protein expression, but this has been challenging to do with standard western blotting. Pan et al. describe a functionalized, water-soluble nanopolymer reagent dubbed pIMAGO, which is used in conjunction with an antibody to measure protein phosphorylation levels with high quantitative accuracy; values are then normalized by protein concentration. pIMAGO contains titanium(IV) ions to capture phosphoproteins and infrared fluorescent dyes for high sensitivity detection. Applying pIMAGO in a microplate format, Pan et al. monitored spleen tyrosine kinase phosphorylation in breast cancer cell lines.