Biosensor-based micro-affinity purification for the proteomic analysis of protein complexes.

A biosensor-based micro-affinity purification method to recover protein binding partners and their complexes for down stream proteomics analysis has been developed using the BIAcore 3000 fitted with a prototype Surface Prep Unit (SPU). The recombinant GST-intracellular domain of E-cadherin or the recombinant GST−β-catenin binding domain of Adenomatous Polyposis Coli (APC) were immobilized onto the SPU and used to affinity purify binding partners from chromatographically enriched SW480 colon cancer cell lysates. A GST- immobilized surface was used as a control. Samples recovered from the SPU were subjected to SDS-PAGE with sensitive Coomassie staining followed by automated in-gel digestion and LC−MS/MS. The results obtained using the SPU were compared with similar experiments performed using Sepharose beads. Keywords: biosensor micro-affinity purification • proteomics • APC • E-cadherin • β-catenin