Simple protein complex purification and identification method for high-throughput mapping of protein interaction networks |
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Authors: | Markillie Lye Meng Lin Chiann-Tso Adkins Joshua N Auberry Deanna L Hill Eric A Hooker Brian S Moore Priscilla A Moore Ronald J Shi Liang Wiley H Steven Kery Vladimir |
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Institution: | Pacific Northwest National Laboratory, 902 Battelle Blvd., P.O. Box 999, Richland, Washington 99354, USA. |
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Abstract: | 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. |
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