Magnetic nanoparticles for protein-protein interaction studies

The analysis of protein complexes and of protein interaction networks is a significant endeavor as nearly all biological processes involve regulated cooperation between multiple protein species in both time and space. Magnetic nanoparticles (NPs) may be used for studies in this field, resorting to a two-step process that involves the tagging or labeling of the desired biological entity with the particle, and the following separation of these tagged entities via mild fluid-based magnetic separation. This work aims at demonstrating the potential of magnetic NPs coated with specific “bait” proteins for addressing a number of reversible molecular interactions, such as protein-protein, antigen-antibody, and enzyme reactions. Nano-sized probes were prepared by conjugating analytical-grade trypsin to conveniently activated magnetic NPs, and used to capture Kunitz-type trypsin inhibitor (KSTI) from a pure protein solution. After isolation of the interacting bait/prey couples (linked to the NPs), the resulting complexes were dissociated and the prey protein was identified by mass spectrometry, giving evidence of the peculiar binding between trypsin and its specific interactor. Anti-gliadin immunoglobulins were also conjugated to NPs and used to recover gliadins according to the same approach. Immunoprecipitation experiments, followed by SDS-PAGE analysis revealed that gliadins specifically interacted with their antibodies. These data indicate that our conjugation procedure does not impair the structure of proteins and the interactions with their appropriate biological systems. Moreover, pending investigation on more complex systems where the specific protein may eventually be present in traces, this exploratory study paves the way to use of these conjugates as nano-sized probes to separate biological entities from their native environment for their identification, quantitation, and further characterization.