Protein-protein interaction (PPI) analysis
This tends to fall into a few different categories:
- Affinity-Purification mass spectrometry (AP-MS): Requires an affinity tagged bait (3xFLAG, Strep, GFP, etc.).
- Endogenous proteins immunoprecipitation: When you are only interested in identifying or quantifying interaction partners for a single protein and you have a good antibody for that protein.
- Proximity-Labeling MS: Relies on fusion of a labeling enzyme (APEX, BirA, etc.) to your protein of interest. Labels everything in ~10-20nm proximity of your fusion protein, but direct binding proteins, as well as those only in spatial proximity. Can be better for identifying transient interaction partners, at the cost of decreased specificity.
In any of these contexts experimental design is critical. The average "purification" results in the the identification of 500-2000 proteins by MS analysis - the vast majority of which are background proteins binding to your beads. As such, having controls that well represent your background is critical. These can include one or several options, such as:
- empty vector purification
- GFP along
- spatial reference proteins (i.e. proteins you expect to reside in similar cellular localization, but not within the same protein complex).