Protein-protein interactions are of great value in biology as they place proteins in a functional context. Interactomics has long been a cornerstone of the proteomics tool chest and is routinely performed in laboratories worldwide. Based on a streamlined and high sensitivity technology capable of acquiring 60 interactomes per day, we recently reported the first substantially complete interactome of an organism – baker"s yeast. We found that almost all proteins are "social" meaning that they have at least one interaction partner and that the mean degree of connection between proteins is surprisingly similar to that in human social networks.
Here we report on our efforts to further scale up and optimize out interactomics workflow. We compare the timsTOF and the new Orbitrap Astral platforms for this purpose. We also investigate different affinity tags, including the recently introduced ALFA tag which is a new, very short epitope that is rationally designed to form a stable alpha helix, against which a highly optimized nanobody with low picomolar affinity exists. We test and compare custom made nanobody-coated plates for this purpose. Furthermore, we take advantage of even faster Evosep gradients to increase the number of interactomes to at least 120 per day, thereby doubling the already high throughput. This opens up the capability to acquire highly standardized interactomes as a function of various perturbations.
We apply our improved interactomics technology to a variety of human cellular systems. We are particularly interested in the interaction of a broad range of viral factors with the human intracellular host machinery.