Unbiased identification of protein-bait interactions using biochemical enrichment and quantitative proteomics

Abstract

The use of recombinant proteins, antibodies, small molecules, or nucleic acids as affinity reagents is a simple yet powerful strategy to study the protein-bait interactions that drive biological processes. However, such experiments are often analyzed by Western blotting, limiting the ability to detect novel protein interactors. Unbiased protein identification by mass spectrometry (MS) extends these experiments beyond the study of pairwise interactions, allowing analyses of whole networks of protein-bait interactions. With the latest advances in MS, it is not uncommon to identify thousands of proteins from complex mixtures. Paradoxically, the improved sensitivity of proteomic analyses can make it more difficult to distinguish bait-specific interactions from the large background of identified proteins. In quantitative proteomics, MS signals from protein populations labeled with stable isotopes such as (13)C and (15)N can be identified and quantified relative to unlabeled counterparts. Using quantitative proteomics to compare biochemical enrichments with the bait of interest against those obtained with control baits allows sensitive detection and discrimination of specific protein-bait interactions among the large number of nonspecific interactions with beads. Ad hoc optimization of enrichment conditions is minimized, and mild purification conditions preserve secondary or high-order protein-protein interactions. The combination of biochemical enrichment and quantitative proteomics allows rapid characterization of molecular baits with their interacting proteins, providing tremendous insight into their biological mechanisms of action.