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 re...

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Bibliographic Details
Published in:Journal of proteome research 2005-09, Vol.4 (5), p.1646-1656
Main Authors: Catimel, B, Rothacker, J, Catimel, J, Faux, M, Ross, J, Connolly, L, Clippingdale, A, Burgess, A. W, Nice, E
Format: Article
Language:English
Subjects:
beta Catenin - chemistry
Biosensing Techniques
Blotting, Western
Cadherins - chemistry
Cell Line, Tumor
Chromatography
Chromatography, Ion Exchange
Chromatography, Liquid
Electrophoresis, Polyacrylamide Gel
Glutathione Transferase - metabolism
Humans
Mass Spectrometry
Peptides - chemistry
Phosphorylation
Protein Binding
Protein Structure, Tertiary
Proteins - chemistry
Proteomics - methods
Recombinant Proteins - chemistry
Sepharose - pharmacology
Time Factors
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Summary: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
ISSN:1535-3893
1535-3907
DOI:10.1021/pr050132x