The bottleneck of interactome analysis: comparison of different ways to treat coimmunoprecipitated complexes

The interactome analysis relies on robust, reliable, reproducible sample preparation to capture the interacting protein partners. At the same time, the sample should be free of contaminants, the proteins unrelated to the studied protein complex. Therefore, balancing the stringency of conditions is essential to minimize background noise while preserving weak interactions. In this context, we evaluated the effectiveness of various procedures typically employed at the final stage of interactome capture before protein digestion.

In the presented work we compared several approaches for processing co-immunoprecipitated protein complexes on affinity magnetic beads. We typically use direct digestion on the beads for this task, but it may lead to the loss of weak interactions due to washes required to remove detergents after the pull-down. Thus, we modified the washing procedure to capture all interactors. In addition, we have modified the conditions for the release of antigen-antibody binding either by competitive binder or by efficient wash conditions to be able to release the protein complexes and not need to deal with antibody peptides. We focused on Transitional endoplasmic reticulum ATPase (VCP) protein complexes with well-characterized protein interactors for the procedure performance testing. This protein is present in the nucleus and cytosol, including many subcellular compartments. Its known interacting partners range from strong binders to weak and transient interactors. Therefore, it represents an optimal model to study the best approach for capturing a wide spectrum of the interaction network. This protein plays an important role also in host-pathogen interaction and is known to be involved in the virus-cell cycle of the Hepatitis B Virus.

The sample digestion was performed in several replicates, applied through a traditional LC-MS pipeline ending on Orbitrap Fusion LUMOS, and data were searched, processed, and statistically evaluated.