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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References
Brockhurst, M.A., Colegrave, N. & Rozen, D.E. Next-generation sequencing as a tool to study microbial evolution. Mol. Ecol. 20, 972–980 (2011).
Ross, J.S. & Cronin, M. Whole cancer genome sequencing by next-generation methods. Am. J. Clin. Pathol. 136, 527–539 (2011).
Charbonnier, S., Gallego, O. & Gavin, A.-C. The social network of a cell: recent advances in interactome mapping. Biotechnol. Annu. Rev. 14, 1–28 (2008).
Collins, M.O. & Choudhary, J.S. Mapping multiprotein complexes by affinity purification and mass spectrometry. Curr. Opin. Biotechnol. 19, 324–330 (2008).
Kiemer, L. & Cesareni, G. Comparative interactomics: comparing apples and pears? Trends Biotechnol. 25, 448–454 (2007).
Williamson, M.P. & Sutcliffe, M.J. Protein-protein interactions. Biochem. Soc. Trans. 38, 875–878 (2010).
del Sol, A., Balling, R., Hood, L. & Galas, D. Diseases as network perturbations. Curr. Opin. Biotechnol. 21, 566–571 (2010).
Stumpf, M.P.H. et al. Estimating the size of the human interactome. Proc. Natl. Acad. Sci. USA 105, 6959–6964 (2008).
Menche, J. et al. Disease networks. Uncovering disease-disease relationships through the incomplete interactome. Science 347, 1257601 (2015).
LaCava, J. et al. Affinity proteomics to study endogenous protein complexes: pointers, pitfalls, preferences and perspectives. Biotechniques 58, 103–119 (2015).
Bell, A.W., Nilsson, T., Kearney, R.E. & Bergeron, J.J.M. The protein microscope: incorporating mass spectrometry into cell biology. Nat. Methods 4, 783–784 (2007).
Devos, D. & Russell, R.B. A more complete, complexed and structured interactome. Curr. Opin. Struct. Biol. 17, 370–377 (2007).
Breitkreutz, B.-J. et al. The BioGRID interaction database: 2008 update. Nucleic Acids Res. 36, D637–D640 (2008).
Armean, I.M., Lilley, K.S. & Trotter, M.W.B. Popular computational methods to assess multiprotein complexes derived from label-free affinity purification and mass spectrometry (AP-MS) experiments. Mol. Cell. Proteomics 12, 1–13 (2013).
Pardo, M. & Choudhary, J.S. Assignment of protein interactions from affinity purification/mass spectrometry data. J. Proteome Res. 11, 1462–1474 (2012).
Trinkle-Mulcahy, L. et al. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J. Cell Biol. 183, 223–239 (2008).
Mellacheruvu, D. et al. The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat. Methods 10, 730–736 (2013).
Babu, M. et al. Interaction landscape of membrane-protein complexes in Saccharomyces cerevisiae. Nature 489, 585–589 (2012).
Jancarik, J. & Kim, S.-H. Sparse matrix sampling: a screening method for crystallization of proteins. J. Appl. Crystallogr. 24, 409–411 (1991).
Chayen, N.E. High-throughput protein crystallization. Adv. Protein Chem. Struct. Biol. 77, 1–22 (2009).
Oeffinger, M. et al. Comprehensive analysis of diverse ribonucleoprotein complexes. Nat. Methods 4, 951–956 (2007).
Domanski, M. et al. Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels. Biotechniques 0, 1–6 (2012).
Alber, F. et al. The molecular architecture of the nuclear pore complex. Nature 450, 695–701 (2007).
Machesky, L.M. & Gould, K.L. The Arp2/3 complex: a multifunctional actin organizer. Curr. Opin. Cell Biol. 11, 117–121 (1999).
Moseley, J.B. & Goode, B.L. The yeast actin cytoskeleton: from cellular function to biochemical mechanism. Microbiol. Mol. Biol. Rev. 70, 605–645 (2006).
Liu, S.-L., Needham, K.M., May, J.R. & Nolen, B.J. Mechanism of a concentration-dependent switch between activation and inhibition of Arp2/3 complex by coronin. J. Biol. Chem. 286, 17039–17046 (2011).
Duncan, M.C., Cope, M.J., Goode, B.L., Wendland, B. & Drubin, D.G. Yeast Eps15-like endocytic protein, Pan1p, activates the Arp2/3 complex. Nat. Cell Biol. 3, 687–690 (2001).
Moreau, V., Galan, J.M., Devilliers, G., Haguenauer-Tsapis, R. & Winsor, B. The yeast actin-related protein Arp2p is required for the internalization step of endocytosis. Mol. Biol. Cell 8, 1361–1375 (1997).
Tang, H.Y., Munn, A. & Cai, M. EH domain proteins Pan1p and End3p are components of a complex that plays a dual role in organization of the cortical actin cytoskeleton and endocytosis in Saccharomyces cerevisiae. Mol. Cell. Biol. 17, 4294–4304 (1997).
Mitchell, P. et al. Rrp47p is an exosome-associated protein required for the 3′ processing of stable RNAs. Mol. Cell. Biol. 23, 6982–6992 (2003).
Wasmuth, E.V. & Lima, C.D. Structure and activities of the eukaryotic RNA exosome. Enzymes 31, 53–75 (2012).
Allmang, C. et al. The yeast exosome and human PM-Scl are related complexes of 3′→5′ exonucleases. Genes Dev. 13, 2148–2158 (1999).
Dziembowski, A., Lorentzen, E., Conti, E. & Séraphin, B. A single subunit, Dis3, is essentially responsible for yeast exosome core activity. Nat. Struct. Mol. Biol. 14, 15–22 (2007).
Synowsky, S.A., van den Heuvel, R.H.H., Mohammed, S., Pijnappel, P.W.W.M. & Heck, A.J.R. Probing genuine strong interactions and post-translational modifications in the heterogeneous yeast exosome protein complex. Mol. Cell. Proteomics 5, 1581–1592 (2006).
Gottschalk, A. et al. A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins. RNA 4, 374–393 (1998).
Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030–1032 (1999).
Blanc, A., Goyer, C. & Sonenberg, N. The coat protein of the yeast double-stranded-RNA virus L-A attaches covalently to the cap structure of eukaryotic messenger-RNA. Mol. Cell. Biol. 12, 3390–3398 (1992).
Görnemann, J. et al. Cotranscriptional spliceosome assembly and splicing are independent of the Prp40p WW domain. RNA 17, 2119–2129 (2011).
De Craene, J.-O. et al. Rtn1p is involved in structuring the cortical endoplasmic reticulum. Mol. Biol. Cell 17, 3009–3020 (2006).
Dawson, T.R., Lazarus, M.D., Hetzer, M.W. & Wente, S.R. ER membrane-bending proteins are necessary for de novo nuclear pore formation. J. Cell Biol. 184, 659–675 (2009).
Helbig, A.O., Heck, A.J.R. & Slijper, M. Exploring the membrane proteome—challenges and analytical strategies. J. Proteomics 73, 868–878 (2010).
Schuldiner, M. et al. Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell 123, 507–519 (2005).
Creutz, C.E., Snyder, S.L. & Schulz, T.A. Characterization of the yeast tricalbins: membrane-bound multi-C2-domain proteins that form complexes involved in membrane trafficking. Cell. Mol. Life Sci. 61, 1208–1220 (2004).
Tackett, A.J. et al. I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions. J. Proteome Res. 4, 1752–1756 (2005).
Manford, A.G., Stefan, C.J., Yuan, H.L., Macgurn, J.A. & Emr, S.D. ER-to-plasma membrane tethering proteins regulate cell signaling and ER morphology. Dev. Cell 23, 1129–1140 (2012).
Voeltz, G.K., Prinz, W.A., Shibata, Y., Rist, J.M. & Rapoport, T.A. A class of membrane proteins shaping the tubular endoplasmic reticulum. Cell 124, 573–586 (2006).
Puig, O. et al. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229 (2001).
Gavin, A.-C. et al. Proteome survey reveals modularity of the yeast cell machinery. Nature 440, 631–636 (2006).
Krogan, N.J. et al. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440, 637–643 (2006).
Sowa, M.E., Bennett, E.J., Gygi, S.P. & Harper, J.W. Defining the human deubiquitinating enzyme interaction landscape. Cell 138, 389–403 (2009).
Behrends, C., Sowa, M.E., Gygi, S.P. & Harper, J.W. Network organization of the human autophagy system. Nature 466, 68–76 (2010).
Ebright, R.H. RNA polymerase: structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II. J. Mol. Biol. 304, 687–698 (2000).
Sukhodolets, M.V., Cabrera, J.E., Zhi, H.J. & Jin, D.J. RapA, a bacterial homolog of SWI2/SNF2, stimulates RNA polymerase recycling in transcription. Genes Dev. 15, 3330–3341 (2001).
Lubas, M. et al. Interaction profiling identifies the human nuclear exosome targeting complex. Mol. Cell 43, 624–637 (2011).
Andersen, P.R. et al. The human cap-binding complex is functionally connected to the nuclear RNA exosome. Nat. Struct. Mol. Biol. 20, 1367–1376 (2013).
Anonymous. The call of the human proteome. Nat. Methods 7, 661 (2010).
Gauci, V.J., Wright, E.P. & Coorssen, J.R. Quantitative proteomics: assessing the spectrum of in-gel protein detection methods. J. Chem. Biol. 4, 3–29 (2011).
Candiano, G. et al. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25, 1327–1333 (2004).
Chambers, M.C. et al. A cross-platform toolkit for mass spectrometry and proteomics. Nat. Biotechnol. 30, 918–920 (2012).
Craig, R. & Beavis, R.C. TANDEM: matching proteins with tandem mass spectra. Bioinformatics 20, 1466–1467 (2004).
Ward, J.H. Jr. Hierarchical grouping to optimize an objective function. J. Am. Stat. Assoc. 58, 236–244 (1963).
Sokal, R.R. & Rohlf, F.J. The comparison of dendrograms by objective methods. Taxon 11, 33–40 (1962).
Pettersen, E.F. et al. UCSF chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Wessel, D. & Flügge, U.I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141–143 (1984).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
R Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2012).
Ellison, A.M. Effect of seed dimorphism on the density-dependent dynamics of experimental populations of Atriplex triangularis (Chenopodiaceae). Am. J. Bot. 74, 1280–1288 (1987).
Statistical Analysis System Institute. SAS/STAT User's Guide (SAS, 1990).
Hartigan, J.A. & Hartigan, P.M. The dip test of unimodality. Ann. Stat. 13, 70–84 (1985).
Akaike, H. A new look at the statistical model identification. IEEE Trans. Automat. Contr. 19, 716–723 (1974).
Benaglia, T., Chauveau, D., Hunter, D.R. & Young, D.S. mixtools: an R package for analyzing finite mixture models. J. Stat. Softw. 32, 1–29 (2009).
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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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.
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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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DOI: https://doi.org/10.1038/nmeth.3395
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