Functional proteomics: protein-protein interactions in vivo.

Abstract

Functional proteomics constitutes an emerging research area in the proteomic field focused to two major targets, the elucidation of biological function of unknown proteins and the definition of cellular mechanisms at the molecular level. Understanding protein functions as well as unravelling molecular mechanisms within the cell is then depending on the identification of the interacting protein partners. The association of an unknown protein with partners belonging to a specific protein complex involved in a particular mechanism would in fact be strongly suggestive of its biological function. Furthermore, a detailed description of the cellular signalling pathways might greatly benefit from the elucidation of protein-protein interactions in the cell. Isolation of functional protein complexes essentially rely on affinity-based procedures. The protein of interest and its specific partners can be fished out from the cellular extract by using a suitable ligand as a bait taking advantage of the specific binding properties of the ligand molecule immobilised on agarose-sepharose supports. Alternative strategies essentially relying on immunoprecipitation techniques have been introduced to allow purification of protein complexes formed in vivo within the cell. The gene coding for the bait tagged with an epitope against which good antibodies exist (FLAG, HA, c-myc, etc.), is transfected into the appropriate cell line and expressed in the cognate host. The cell extracts are immunoprecipitated with anti-tag monoclonal antibodies using suitable experimental conditions to avoid dissociation of the complexes. In both cases, protein components specifically recognised by the bait and retained on the agarose beads can then be eluted and fractionated by SDS-PAGE. The protein bands detected on the gel are in situ enzymatically digested and the resulting peptide mixtures analysed by capillary LC-MS/MS techniques leading to the identification of the protein interactors.