Bait Correlation Improves Interactor Identification by Tandem Mass Tag-Affinity Purification-Mass Spectrometry.

Autor:	Mei L; Department of Chemistry, University of California, Riverside, California 92521, United States., Montoya MR; Department of Chemistry, University of California, Riverside, California 92521, United States., Quanrud GM; Department of Chemistry, University of California, Riverside, California 92521, United States., Tran M; Department of Chemistry, University of California, Riverside, California 92521, United States., Villa-Sharma A; Department of Chemistry, University of California, Riverside, California 92521, United States., Huang M; Environmental Toxicology Graduate Program, University of California, Riverside, California 92521, United States., Genereux JC; Department of Chemistry, University of California, Riverside, California 92521, United States.; Environmental Toxicology Graduate Program, University of California, Riverside, California 92521, United States.
Jazyk:	angličtina
Zdroj:	Journal of proteome research [J Proteome Res] 2020 Apr 03; Vol. 19 (4), pp. 1565-1573. Date of Electronic Publication: 2020 Mar 25.
DOI:	10.1021/acs.jproteome.9b00825
Abstrakt:	The quantitative multiplexing capacity of isobaric tandem mass tags (TMT) has increased the throughput of affinity purification mass spectrometry (AP-MS) to characterize protein interaction networks of immunoprecipitated bait proteins. However, variable bait levels between replicates can convolute interactor identification. We compared the Student's t -test and Pearson's R correlation as methods to generate t -statistics and assessed the significance of interactors following TMT-AP-MS. Using a simple linear model of protein recovery in immunoprecipitates to simulate reporter ion ratio distributions, we found that correlation-derived t- statistics protect against bait variance while robustly controlling type I errors (false positives). We experimentally determined the performance of these two approaches for determining t -statistics under two experimental conditions: irreversible prey association to the Hsp40 mutant DNAJB8 H31Q followed by stringent washing, and reversible association to 14-3-3ζ with gentle washing. Correlation-derived t -statistics performed at least as well as Student's t -statistics for each sample and with substantial improvement in performance for experiments with high bait-level variance. Deliberately varying bait levels over a large range fails to improve selectivity but does increase the robustness between runs. The use of correlation-derived t -statistics should improve identification of interactors using TMT-AP-MS. Data are available via ProteomeXchange with identifier PXD016613.
Databáze:	MEDLINE
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