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Simple Protein Complex Purification and Identification Method for High-Throughput Mapping of Protein Interaction Networks

2005; American Chemical Society; Volume: 4; Issue: 2 Linguagem: Inglês

10.1021/pr049847a

1535-3907

Lye Meng Markillie, Chiann‐Tso Lin, Joshua Adkins, Deanna L. Auberry, Eric A. Hill, Brian S. Hooker, Priscilla A. Moore, Ronald J. Moore, Liang Shi, H Wiley, Vladimír Kéry,

Biotin and Related Studies

Most current methods for purification and identification of protein complexes use endogenous expression of affinity-tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, and gel separation and in-gel digestion prior to mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pull-down assay with denaturing elution, trypsin digestion in organic solvent, and LC−ESI MS/MS protein identification using SEQUEST analysis. Our method is simple and easy to scale-up and automate, making it suitable for high-throughput mapping of protein interaction networks and functional proteomics. Keywords: protein complex purification • identification • mass spectrometry • mapping protein interaction networks