Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks

29.07.2011

Deswal S, Schulze AK, Höfer T, Schamel WW

PLoS One 6(7):e22928

PLoS One        online article

For a detailed understanding of signal transduction pathways it is helpful to generate mathematical models to describe them. To do so, it is necessary to generate dynamic and quantitative data sets. Hence, it is vital to develop  methods which can generate very accurate data on protein interactions and phosphorylations. In this BIOSS publication, we have developed a method called multi-colour ImmunoPrecipitation measured by Flow CytoMetry (IP-FCM), where antibody-coupled latex beads capture the protein of interest from cellular lysates and are then stained with differently fluorescent-labelled antibodies to quantify the amount of the immunoprecipitated protein, of an interaction partner and of phosphorylation sites. The fluorescence is measured using flow cytometry. Using IP-FCM, we derived quantitative data on the membrane-proximal T-cell antigen receptor (TCR-CD3) signaling network, including the recruitment of the kinase ZAP70 to the TCR-CD3 and subsequent ZAP70 activation by phosphorylation, which was used to build mathematical model. The accuracy of IP-FCM was found to be 1o times higher than IP-Western Blotting, thus IP-FCM offers the opportunity to generate suitable data for mathematical modeling and understanding of  signaling networks.