Abstract
We have developed an original experimental approach based on the use of surface plasmon resonance (SPR) biosensors, applicable for investigation of potential partners involved in protein–protein interactions (PPI) as well as protein–peptide or protein–small molecule interactions. It is based on combining a SPR biosensor, size exclusion chromatography (SEC), mass spectrometric identification of proteins (LC-MS/MS) and direct molecular fishing employing principles of affinity chromatography for isolation of potential partner proteins from the total lysate of biological samples using immobilized target proteins (or small non-peptide compounds) as ligands. Applicability of this approach has been demonstrated within the frame of the Human Proteome Project (HPP) and PPI regulation by a small non-peptide biologically active compound, isatin.
Highlights
There is increasing evidence that in living systems proteins exist and function within stable or dynamic molecular complexes [1]
Direct molecular fishing is based on the affinity interaction of partner proteins present in the lysate of the biological material with the immobilized target bait protein
We have developed an original experimental approach employing surface plasmon resonance (SPR) biosensors and applicable for investigation of potential partners involved in protein–protein as well as protein–peptide or protein–small molecule interactions
Summary
There is increasing evidence that in living systems proteins exist and function within stable or dynamic molecular complexes [1]. Protein–protein interactions (PPIs) determining formation and lifespan of such complexes attract much interest; they are extensively studied by using various bioinformatic, genomic, and biochemical technologies [2,3,4]. Molecular fishing is a variant of affinity-based isolation of target proteins from a lysate of the biological material due to specific interaction between the immobilized ligand (a bait molecule) and its putative (one or several) functionally competent partners (pray molecules) [4,5,6]. We have summarized results of our studies on the use of SPR-based approach for direct molecular fishing of proteins from lysates of biological materials and identification of prey proteins by mass spectrometry. We demonstrate applicability of the SPR biosensor technology for analysis of ligand protein interactions using non-peptide small molecules as baits
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