Many cellular processes are depending on the interplay of different protein complexes, bringing specialized functions in a timely fashion together. Interactomics tries mapping the different interactions of proteins and creating an overall map to understand the mechanisms behind biological functions. Interactome changes can be triggered by changes in post-translational modifications or the special separation of proteins in a cell.
In recent years several new technologies for the mapping of protein interactions have been developed. Many of these technologies are based on affinity purification steps or the introduction of labels that allow the special or timely changes, combined with mass spectrometric methods for the identification of the associated proteins.
This technique is based on the antibody-based enrichment of the protein of interest. With the pull-out of the protein of interest, all interaction partners are co-precipitating, which are then identified by mass spectrometry-based proteomics. Based on the identified proteins, an interaction network can be constructed and further probed for the underlying mechanisms.
Affinity proteomics is hampered by the occurrence of unspecific interactions, which are based on proteins interacting with the beads etc. during the pull-down. An alternative is the in vivo labelling of proteins using different enzymatic reactions. The BioID and TurboID techniques are based on the unspecific labeling of the surrounding proteins with Biotin, which can be used as a covalent handle for the isolation of proteins. The advantage of these methods is the lower background of unspecific interactions. The APEX technique allows the identification of interaction partners using the labelling by a biotin-phenol compound, which is triggered by the presence of H2O2 radicals. The reaction radius is small enough the label only proteins in the direct surroundings of the protein of interest. The Biotin-label can then be used for proteomics or microscopic analysis.
All of the above-described methods are unable to identify specific binding sites within a protein. The interactions are only detected without the possibility to show the specific interaction site. The Prisma techniques, developed in this laboratory, is able to identify the specific interactions site in a protein of interest. For this, the protein of interest is broken down into small peptides, tiling the entire proteins. The resulting peptide matrix is chemically synthesized on a membrane. The membrane is then used to pull out all proteins interacting with the protein of interest. Each of the peptide spots on the membrane is then analyzed by mass spectrometry generating a large data set with all interactions of the protein of interest.
In addition to the special separation of the interaction sites, the Prisma technique allows the measurement of PTM-induced changes to the interactome. The PTM-modified peptides are included in the interaction matrix, and the changes in the binding can be measured by mass spectrometry.
Shown is the interaction screen for the transcription factor C/EBPß, which is a transcription factor involved in many cellular processes, including immune and inflammatory responses. Among other interactions, the Prisma analysis revealed that the interaction of TLE3 is depended on the methylation of C/EBPß. Only the methylated form of C/EBPß is able to bind, which is important for the regulation of fat cell differentiation.