Fig. 1

MAC-tag-based workflow for identification of protein complexes and interactions. Gateway compatible MAC-tag destination vectors containing StrepIII, HA and BirA* were designed to allow the gene of interest either C-terminal or N-terminal tagging. The expression vector can then be transfected into Flp-In T-REx 293 to establish the transgenic stably and inducible expressing isogenic cell lines. For the AP-MS and BioID analysis approaches, the cell line is separated into two cultures, BioID cells receiving addition of 50 μM biotin in their culture medium. In the following protein extraction process, optimized lysis and affinity purification conditions for both analysis approaches were used. The interacting proteins were then analyzed by quantitative mass spectrometry and high-confidence interaction proteins (HCIPs) were inferred via stringent statistical filtering. This integrated workflow allows laborless generation of cellular material for analyses, and results in integrated view of the formed protein complexes, protein-protein interactions and detailed molecular context definition