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The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (K d  values). In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST) we were able to validate interactions and to establish a binding hierarchy based on K d  values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and  in vitro  phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2’s were subjected to MST to determine K d  values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, K d  values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis  in vivo , this work marks the first K d  values for phosphotyrosine/SH2 interactions on a phosphoprotein level.

Completion of Proteomic Data Sets by Kd Measurement Using Cell-Free Synthesis of Site-Specifically Labeled Proteins