Showing papers by "W. Hayes McDonald published in 2007"
TL;DR: A Gateway-compatible dual-tag affinity purification system that integrates regulatable expression, tetracysteine motifs, and various combinations of affinity tags to facilitate the cloning, detection, and purification of bait proteins and their interacting partners is described.
Abstract: Although affinity purification coupled with mass spectrometry (MS) provides a powerful tool to study protein-protein interactions, this strategy has encountered numerous difficulties when adapted to mammalian cells. Here we describe a Gateway-compatible dual-tag affinity purification system that integrates regulatable expression, tetracysteine motifs, and various combinations ofaffinity tags to facilitate the cloning, detection, and purification of bait proteins and their interacting partners. Utilizing the human telomere binding protein TRF2 as a benchmark, we demonstrate bait protein recoveries upwards of approximately 16% from as little as 1-7 x 10(7) cells and successfully identify known TRF2 interacting proteins, suggesting that our dual-tag affinity purification approach is a capable new tool for expanding the capacity to explore mammalian proteomic networks.
33 citations
TL;DR: A general, robust statistical method for inferring authentic interactions from protein prey-by-bait frequency tables using a binomial-based likelihood ratio test (LRT) coupled with Bayes' Odds estimation, and found that Monte Carlo simulation is a feasible tool for checking modeling assumptions, estimating parameters, and evaluating the significance of results in protein association studies.
Abstract: Affinity isolation of protein complexes followed by protein identification by LC−MS/MS is an increasingly popular approach for mapping protein interactions. However, systematic and random assay errors from multiple sources must be considered to confidently infer authentic protein−protein interactions. To address this issue, we developed a general, robust statistical method for inferring authentic interactions from protein prey-by-bait frequency tables using a binomial-based likelihood ratio test (LRT) coupled with Bayes' Odds estimation. We then applied our LRT-Bayes' algorithm experimentally using data from protein complexes isolated from Rhodopseudomonas palustris. Our algorithm, in conjunction with the experimental protocol, inferred with high confidence authentic interacting proteins from abundant, stable complexes, but few or no authentic interactions for lower-abundance complexes. The algorithm can discriminate against a background of prey proteins that are detected in association with a large numbe...
11 citations