We must reliably map the interactomes of cellular macromolecular complexes in order to fully explore and understand biological systems. However, there are no methods to accurately predict how to capture a given macromolecular complex with its physiological binding partners. Here, we present a screening method that comprehensively explores the parameters affecting the stability of interactions in affinity-captured complexes, enabling the discovery of physiological binding partners in unparalleled detail. We have implemented this screen on several macromolecular complexes from a variety of organisms, revealing novel profiles for even well-studied proteins. Our approach is robust, economical and automatable, providing inroads to the rigorous, systematic dissection of cellular interactomes.
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We thank The Rockefeller University High Energy Physics Instrument Shop for diligence in custom apparatus design and fabrication; X. Wang for assistance with MS data analysis; and members of the Chait, Jensen and Rout laboratories for help and discussion. I. Poser and A. Hyman (Max Planck Institute of Molecular Biology and Genetics, Dresden) provided the RBM7-LAP cell line. This work was funded by the US National Institutes of Health (NIH) grant nos. U54 GM103511 and P41 GM109824 (J.D.A., B.T.C. and M.P.R.), P50 GM076547 (J.D.A.) and P41 GM103314 (B.T.C.); the Lundbeck Foundation (to T.H.J. and J.L.) and the Danish National Research Foundations (to T.H.J.).
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Integrated supplementary information
(a) Preparing to use the powder dispensing manifold; pre-cooling with liquid N2; (b) adjustable volume dispensing manifold, shown bottom up; (c) dispensing manifold with 96-well deep-well plate atop, cell material transfer is achieved upon inversion of this assembly; (d) a 96-well filtration device atop a 96-well, deep well collection plate.
Coomassie stained SDS-polyacrylamide gels of Nup1p-SpA screen. Numbers above each lane indicate the extractant formulation, presented in Supplementary Table 1.
SDS-PAGE and LC-MS/MS clustering analysis of Nup1p-Spa 96-well purification. Numbers below each lane indicate the extractant formulation, presented in Supplementary Table 1.
Frequency distribution of correlation coefficients between the gel dendrogram and 10 million permutations of the MS dendrogram.
Coomassie stained SDS-polyacrylamide gels of Arp2p-GFP screen. Numbers above each lane indicate the extractant formulation, presented in Supplementary Table 1. Reference condition # 64.
Coomassie stained SDS-polyacrylamide gels of Csl4p-TAP screen. Numbers above each lane indicate the extractant formulation, presented in Supplementary Table 1. Reference condition # 65.
Coomassie stained SDS-polyacrylamide gels of Snu71p-TAP screen. Numbers above each lane indicate the extractant formulation, presented in Supplementary Table 1. Reference condition # 33.
Coomassie stained SDS-polyacrylamide gels of Rtn1p-GFP screen. Numbers above each lane indicate the extractant formulation, presented in Supplementary Table 1.
Coomassie stained SDS-polyacrylamide gel of RpoC-SpA screen. Numbers above each lane indicate the extractant formulation, presented in Supplementary Table 1.
Coomassie stained SDS-polyacrylamide gel of RRP6-3xFLAG screen. Numbers above each lane indicate the extractant formulation, presented in Supplementary Table 1.
Coomassie stained SDS-polyacrylamide gels of RBM7-LAP screens. Numbers above each lane indicate the extractant formulation, presented in Supplementary Table 1.
The manifold is constructed of Black Delrin (acetal) that tolerates liquid nitrogen temperatures; additional engineering diagrams with more detailed specifications are available upon request.
For 96-well screens with yeast, extract homogenization is assisted by vortexing in the presence of 2 mm Ø steel balls. This manifold provides for parallel dispensing of precisely 2 balls to each well. When placed atop a 96-well plate, removal of the sliding bottom allows the balls to be deposited into the wells of the plate.
Q-Q plot of the measured I-DIRT ratios (normalized to 100%) quantiles vs. theoretical quantiles.
Supplementary Figure 15 Fitted bimodal distribution of I-DIRT ratios of proteins copurifying with Rtn1p.
Orange and blue solid lines – fitted curves; dashed line – kernel density estimate of the total distribution; histogram – frequency distribution of I-DIRT ratios.
Supplementary Figures 1–15, Supplementary Tables 1, 3 and 5, Supplementary Notes 1 and 2, Supplementary Data and Supplementary Protocol 1 (PDF 10634 kb)
LC-MS/MS data of Nup1p-SpA affinity capture (XLSX 418 kb)
MS data for Rtn1p affinity capture experiments (XLSX 1169 kb)
Program files for Hamilton STAR liquid handling workstation (ZIP 88 kb)
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Hakhverdyan, Z., Domanski, M., Hough, L. et al. Rapid, optimized interactomic screening. Nat Methods 12, 553–560 (2015). https://doi.org/10.1038/nmeth.3395
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