Light-controlled affinity purification of protein complexes exemplified by the resting ZAP70 interactome

Front Immunol.           online article

In BIOSS we study intracellular signalling pathways, that are controlled by multiprotein complexes. To identify those complexes, methods to affinity purify them and to determine their composition by mass spectrometry is widely used. One drawback of these methods is the presence of false positives. The elution of the protein of interest is achieved by changing the biochemical properties of the buffer, so that unspecifically bound proteins (the false positives) may also elute. Here, we developed an optogenetics-derived and light-controlled affinity purification method based on the light-regulated reversible protein interaction between phytochrome B (PhyB) and ist phytochrome interacting factor 6 (PIF6). In this system elution is done by changing the wavelength of light, thus reducing false positives. As proof-of-concept, we expressed PIF-tagged variants of the tyrosine kinase ZAP70 in human T cells, purified ZAP70 and associating proteins using our light-controlled system, and identified the interaction partners by quantitative mass spectrometry. Using unstimulated T cells, we were able to detect the known interaction partners, and could filter out all other proteins.