High-Efficiency Enrichment by Saturating Nanoliters of Protein Affinity Media

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.