Rapid, optimized interactomic screening

Abstract

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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Figure 1: Schematic representation of the parallelized affinity capture procedure.
Figure 2: Extraction condition design and copurification pattern analysis.
Figure 3: Nuclear pore complex (NPC) purification from single proteins to macromolecular assemblies.
Figure 4: Affinity capture strategy implementation on different protein complexes, affinity tags and model organisms.
Figure 5: In-depth analysis of Rtn1p affinity capture.

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Acknowledgements

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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Authors

Contributions

J.L. and M.P.R. conceived the screening strategy; J.L. carried out proof-of-concept experiments, assisted by L.E.H.; L.E.H. and V.S. designed manifolds, which were fabricated by V.S. and tested by L.E.H., J.L. and Z.H.; filters were designed by A.A.O., A.R.O. and J.L., fabricated by A.A.O. and A.R.O., and tested by J.L. and Z.H.; J.L., Z.H. and M.D. designed experiments, executed screens and further developed procedures—with yeast work primarily carried out by Z.H. and human cell line work primarily carried out by M.D.; MS analyses were carried out by J.L., Z.H. and K.R.M., with I-DIRT done by Z.H.; transposing the procedure to robotic automation was carried out by D.J.D. assisted by J.L.; J.L., Z.H. and D.F. conceived of the protein copurification gel database and software, which was built by S.K. and D.F. with testing and feedback from J.L. and Z.H.; J.D.A., D.F., B.T.C., T.H.J., M.P.R. and J.L. supervised the project; Z.H., B.T.C., M.P.R. and J.L. wrote the paper.

Corresponding authors

Correspondence to Michael P Rout or John LaCava.

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Competing interests

A.A.O., A.R.O., M.P.R. and J.L. are inventors on a US patent application encompassing the filter work described in this article.

Integrated supplementary information

Supplementary Figure 1 Photographs of the powder-dispensing manifold and filter plate.

(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.

Supplementary Figure 2 Nup1p-SpA 96-well screen.

Coomassie stained SDS-polyacrylamide gels of Nup1p-SpA screen. Numbers above each lane indicate the extractant formulation, presented in Supplementary Table 1.

Supplementary Figure 3 Comparison of SDS-PAGE and direct-to-MS analyses.

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.

Supplementary Figure 4 Correlation of SDS-PAGE and direct-to-MS analyses.

Frequency distribution of correlation coefficients between the gel dendrogram and 10 million permutations of the MS dendrogram.

Supplementary Figure 5 Arp2p-GFP 32-well screen.

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.

Supplementary Figure 6 Csl4p-TAP 32-well screen.

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.

Supplementary Figure 7 Snu71p-TAP 32-well screen.

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.

Supplementary Figure 8 Rtn1p-GFP 32-well screen.

Coomassie stained SDS-polyacrylamide gels of Rtn1p-GFP screen. Numbers above each lane indicate the extractant formulation, presented in Supplementary Table 1.

Supplementary Figure 9 RpoC-SpA 24-well screen.

Coomassie stained SDS-polyacrylamide gel of RpoC-SpA screen. Numbers above each lane indicate the extractant formulation, presented in Supplementary Table 1.

Supplementary Figure 10 RRP6-3×Flag 24-well screen.

Coomassie stained SDS-polyacrylamide gel of RRP6-3xFLAG screen. Numbers above each lane indicate the extractant formulation, presented in Supplementary Table 1.

Supplementary Figure 11 RBM7-LAP two 24-well screens.

Coomassie stained SDS-polyacrylamide gels of RBM7-LAP screens. Numbers above each lane indicate the extractant formulation, presented in Supplementary Table 1.

Supplementary Figure 12 Dispensing manifold, basic engineering diagram.

The manifold is constructed of Black Delrin (acetal) that tolerates liquid nitrogen temperatures; additional engineering diagrams with more detailed specifications are available upon request.

Supplementary Figure 13 Bead-dispensing manifold.

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.

Supplementary Figure 14 Testing normality of I-DIRT ratio distribution.

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 information

Supplementary Text and Figures

Supplementary Figures 1–15, Supplementary Tables 1, 3 and 5, Supplementary Notes 1 and 2, Supplementary Data and Supplementary Protocol 1 (PDF 10634 kb)

Supplementary Table 2

LC-MS/MS data of Nup1p-SpA affinity capture (XLSX 418 kb)

Supplementary Table 4

MS data for Rtn1p affinity capture experiments (XLSX 1169 kb)

Supplementary Protocol 2

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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