Protein interactomics based on direct molecular fishing on paramagnetic particles: Experimental simulation and SPR validation

Abstract

We describe an experimental approach for direct molecular fishing of prey protein on the surface of two types of paramagnetic particles (PMP) having different size and composition. Human microsomal cytochrome b(5) (b(5)) and its known partner human cytochrome P450 3A5 (CYP3A5) were used as bait and prey proteins, respectively. For assessing the level of unspecific binding of background proteins, α-fetoprotein (aFP) was used. SPR measurements were applied for quantitative analysis of trapped proteins (CYP3A5 and aFP) after fishing on PMP. It was shown that the described approach of molecular fishing on micro-PMP provides enough prey proteins for LC-MS/MS identification and SPR validation, so this approach can be used for discovery of new protein-protein interactions in the framework of Human Proteome Project.