High-Efficiency Enrichment by Saturating Nanoliters of Protein Affinity Media.

Abstract

Affinity-purification mass spectrometry (AP-MS) is an established technique for identifying protein-protein interactions (PPIs). The basic technology involves immobilizing a high-specificity ligand to a solid-phase support (e.g., an agarose or magnetic bead) to pull down protein(s) of interest from cell lysates. Although these supports are engineered to minimize interactions with background protein, the conventional method recovers mostly nonspecific binders. The law of mass action for dilute solutions has taught us to use an excess of beads to capture all target proteins, especially weakly interacting ones. However, modern microbead technology presents a binding environment that is much different from a dilute solution. We describe a fluidic platform that captures and processes ultralow nanoliter quantities of magnetic particles, simultaneously increasing the efficiency of PPI detection and strongly suppressing nonspecific binding. We demonstrate the concept with synthetic mixtures of tagged protein and illustrate performance with a variety of AP-MS experiment types. These include a BioID experiment targeting lamin-A interactors from HeLa cells and pulldowns using GFP-tagged proteins associated with a double-strand DNA repair mechanism. We show that efficient extraction requires saturation of the solid-phase support and that <10 nL of beads is sufficient to generate comprehensive protein interaction maps.