SILAC-Pulse Proteolysis: A Mass Spectrometry-Based Method for Discovery and Cross-Validation in Proteome-Wide Studies of Ligand Binding

Research Article

Abstract

Reported here is the use of stable isotope labeling with amino acids in cell culture (SILAC) and pulse proteolysis (PP) for detection and quantitation of protein–ligand binding interactions on the proteomic scale. The incorporation of SILAC into PP enables the PP technique to be used for the unbiased detection and quantitation of protein–ligand binding interactions in complex biological mixtures (e.g., cell lysates) without the need for prefractionation. The SILAC-PP technique is demonstrated in two proof-of-principle experiments using proteins in a yeast cell lysate and two test ligands including a well-characterized drug, cyclosporine A (CsA), and a non-hydrolyzable adenosine triphosphate (ATP) analogue, adenylyl imidodiphosphate (AMP-PNP). The well-known tight-binding interaction between CsA and cyclophilin A was successfully detected and quantified in replicate analyses, and a total of 33 proteins from a yeast cell lysate were found to have AMP-PNP-induced stability changes. In control experiments, the method’s false positive rate of protein target discovery was found to be in the range of 2.1% to 3.6%. SILAC-PP and the previously reported stability of protein from rates of oxidation (SPROX) technique both report on the same thermodynamic properties of proteins and protein–ligand complexes. However, they employ different probes and mass spectrometry-based readouts. This creates the opportunity to cross-validate SPROX results with SILAC-PP results, and vice-versa. As part of this work, the SILAC-PP results obtained here were cross-validated with previously reported SPROX results on the same model systems to help differentiate true positives from false positives in the two experiments.

Graphical Abstract

Key words

Mass spectrometry Proteomics ATP Cyclosporine A Thermodynamics Protein folding Chemical denaturation 

Supplementary material

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Table S1A-C Summary of experimental conditions used in (A) the CsA-Binding Experiments, (B) the ATP-Binding Experiments, and (C) the control experiments (XLSX 13 kb)
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Table S2-S21A and BExcel spreadsheets containing (A) a listing of the peptide and protein ID’s generated in the Control, CsA-, and ATP-Binding Experiments, and (B) a listing of the peptides and proteins assayed in each experiment (XLSX 736 kb)
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Table S22A-CSummary of the protein hits identified in (A) the control experiments, (B) the ATP-binding experiments, and (C) the CsA-binding experiments (XLSX 23 kb)

References

  1. 1.
    West, G.M., Tang, L., Fitzgerald, M.C.: Thermodynamic analysis of protein stability and ligand binding using a chemical modification- and mass spectrometry-based strategy. Anal. Chem. 80(11), 4175–4185 (2008)CrossRefGoogle Scholar
  2. 2.
    West, G.M., Tucker, C.L., Xu, T., Park, S.K., Han, X., Yates III, J.R., Fitzgerald, M.C.: Quantitative proteomics approach for identifying protein–drug interactions in complex mixtures using protein stability measurements. Proc. Natl. Acad. Sci. U. S. A. 107(20), 9078–9082 (2010)CrossRefGoogle Scholar
  3. 3.
    Park, C., Marqusee, S.: Pulse proteolysis: a simple method for quantitative determination of protein stability and ligand binding. Nat. Methods 2(3), 207–212 (2005)CrossRefGoogle Scholar
  4. 4.
    Liu, P.F., Kihara, D., Park, C.: Energetics-based discovery of protein–ligand interactions on a proteomic scale. J. Mol. Biol. 408(1), 147–162 (2011)CrossRefGoogle Scholar
  5. 5.
    Xu, Y., Falk, I.N., Hallen, M.A., Fitzgerald, M.C.: Mass spectrometry- and lysine amidination-based protocol for thermodynamic analysis of protein folding and ligand binding interactions. Anal. Chem. 83(9), 3555–3562 (2011)CrossRefGoogle Scholar
  6. 6.
    Tran, D.T., Banerjee, S., Alayash, A.I., Crumbliss, A.L., Fitzgerald, M.C.: Slow histidine H/D exchange protocol for thermodynamic analysis of protein folding and stability using mass spectrometry. Anal. Chem. 84(3), 1653–1660 (2012)CrossRefGoogle Scholar
  7. 7.
    Ghaemmaghami, S., Fitzgerald, M.C., Oas, T.G.: A quantitative, high-throughput screen for protein stability. Proc. Natl. Acad. Sci. U. S. A. 97(15), 8296–8301 (2000)CrossRefGoogle Scholar
  8. 8.
    Isom, D.G., Vardy, E., Oas, T.G., Hellinga, H.W.: Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity. Proc. Natl. Acad. Sci. U. S. A. 107(11), 4908–4913 (2010)CrossRefGoogle Scholar
  9. 9.
    Dearmond, P.D., Xu, Y., Strickland, E.C., Daniels, K.G., Fitzgerald, M.C.: Thermodynamic analysis of protein-ligand interactions in complex biological mixtures using a shotgun proteomics approach. J. Protein Res. 10(11), 4948–4958 (2011)CrossRefGoogle Scholar
  10. 10.
    Strickland, E.C., Geer, M.A., Tran, D.T., Adhikari, J., West, G.M., DeArmond, P.D., Xu, Y., Fitzgerald, M.C.: Thermodynamic analysis of protein-ligand binding interactions in complex biological mixtures using the stability of proteins from rates of oxidation. Nat. Protoc. 8(1), 148–161 (2013)CrossRefGoogle Scholar
  11. 11.
    Chang, Y., Schlebach, J.P., Verheul, R.A., Park, C.: Simplified proteomics approach to discover protein–ligand interactions. Protein Sci. 21(9), 1269–1277 (2012)CrossRefGoogle Scholar
  12. 12.
    Molina, D.M., Jafari, R., Ignatushchenko, M., Seki, T., Larsson, E.A., Dan, C., Sreekumar, L., Cao, Y., Nordlund, P.: Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341(6141), 84–87 (2013)CrossRefGoogle Scholar
  13. 13.
    Tran, D.T., Adhikari, J., Fitzgerald, M.C.: SILAC-based strategy for proteome-wide thermodynamic analysis of protein-ligand binding interactions. Mol. Cell. Proteomics 13, 1800–1813 (2014)CrossRefGoogle Scholar
  14. 14.
    Strickland, E.C., Geer, M.A., Hong, J.Y., Fitzgerald, M.C.: False-positive rate determination of protein target discovery using a covalent modification- and mass spectrometry-based proteomics platform. J. Am. Soc. Mass Spectrom. 25(1), 132–140 (2014)CrossRefGoogle Scholar
  15. 15.
    Lomenick, B., Olsen, R.W., Huang, J.: Identification of direct protein targets of small molecules. ACS Chem. Biol. 6(1), 34–46 (2011)CrossRefGoogle Scholar
  16. 16.
    Villamor, J.G., Kaschani, F., Colby, T., Oeljeklaus, J., Zhao, D., Kaiser, M., Patricelli, M.P., van der Hoorn, R.A.L.: Profiling protein kinases and other ATP binding proteins in Arabidopsis using Acyl-ATP probes. Mol. Cell. Proteomics 12(9), 2481–2496 (2013)CrossRefGoogle Scholar
  17. 17.
    Lomenick, B., Hao, R., Jonai, N., Chin, R.M., Aghajan, M., Warburton, S., Wang, J.N., Wu, R.P., Gomez, F., Loo, J.A., Wohlschlegel, J.A., Vondriska, T.M., Pelletier, J., Herschman, H.R., Clardy, J., Clarke, C.F., Huang, J.: Target identification using drug affinity responsive target stability (DARTS). Proc. Natl. Acad. Sci. U. S. A. 106(51), 21984–21989 (2009)CrossRefGoogle Scholar
  18. 18.
    Walther, T.C., Olsen, J.V., Mann, M.: Yeast expression proteomics by high-resolution mass spectrometry. Methods Enzymol. 470, 259–280 (2010)CrossRefGoogle Scholar
  19. 19.
    Pace, C.N.: Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 131, 266–280 (1986)CrossRefGoogle Scholar
  20. 20.
    Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V., Mann, M.: In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1(6), 2856–2860 (2006)CrossRefGoogle Scholar
  21. 21.
    Wang, M.Z., Shetty, J.T., Howard, B.A., Campa, M.J., Patz Jr., E.F., Fitzgerald, M.C.: Thermodynamic analysis of cyclosporin a binding to cyclophilin a in a lung tumor tissue lysate. Anal. Chem. 76(15), 4343–4348 (2004)CrossRefGoogle Scholar
  22. 22.
    Myers, J.K., Pace, C.N., Scholtz, J.M.: Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 4(10), 2138–2148 (1995)CrossRefGoogle Scholar
  23. 23.
    Prodromou, C., Roe, S.M., OBrien, R., Ladbury, J.E., Piper, P.W., Pearl, L.H.: Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone. Cell 90(1), 65–75 (1997)CrossRefGoogle Scholar
  24. 24.
    Harding, M.W., Handschumacher, R.E.: Cyclophilin, a primary molecular target for cyclosporine. Structural and functional implications. Transplantation 46(2 Suppl), 29S–35S (1988)CrossRefGoogle Scholar
  25. 25.
    Handschumacher, R.E., Harding, M.W., Rice, J., Drugge, R.J., Speicher, D.W.: Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 226(4674), 544–547 (1984)CrossRefGoogle Scholar
  26. 26.
    Liu, J., Albers, M.W., Chen, C.M., Schreiber, S.L., Walsh, C.T.: Cloning, expression, and purification of human cyclophilin in Escherichia coli and assessment of the catalytic role of cysteines by site-directed mutagenesis. Proc. Natl. Acad. Sci. U. S. A. 87(6), 2304–2308 (1990)CrossRefGoogle Scholar
  27. 27.
    Jorgensen, P.L., Hakansson, K.O., Karlish, S.J.D.: Structure and mechanism of Na,K-ATPase: functional sites and their interactions. Annu. Rev. Physiol. 65, 817–849 (2003)CrossRefGoogle Scholar
  28. 28.
    Arnesano, F., Banci, L., Bertini, I., Ciofi-Baffoni, S., Molteni, E., Huffman, D.L., O Halloran, T.V.: Metallochaperones and metal-transporting ATPases: a comparative analysis of sequences and structures. Genome Res. 12(2), 255–271 (2002)Google Scholar
  29. 29.
    Pause, A., Methot, N., Sonenberg, N.: The HRIGRXXR region of the dead box RNA helicase eukaryotic translation initiation factor-4a is required for RNA-binding and ATP hydrolysis. Mol. Cell. Biol. 13(11), 6789–6798 (1993)Google Scholar
  30. 30.
    Weng, Y.M., Czaplinski, K., Peltz, S.W.: ATP is a cofactor of the Upf1 protein that modulates its translation termination and RNA binding activities. RNA-Publ. RNA Soc. 4(2), 205–214 (1998)Google Scholar
  31. 31.
    DeArmond, P.D., West, G.M., Huang, H.T., Fitzgerald, M.C.: Stable isotope labeling strategy for protein–ligand binding analysis in multi-component protein mixtures. J. Am. Soc. Mass Spectrom. 22(3), 418–430 (2011)CrossRefGoogle Scholar
  32. 32.
    Apweiler, R., Bateman, A., Martin, M.J., O Donovan, C., Magrane, M., Alam-Faruque, Y., Alpi, E., Antunes, R., Arganiska, J., Casanova, E.B., Bely, B., Bingley, M., Bonilla, C., Britto, R., Bursteinas, B., Chan, W.M., Chavali, G., Cibrian-Uhalte, E., Da Silva, A., De Giorgi, M., Fazzini, F., Gane, P., Castro, L.G., Garmiri, P., Hatton-Ellis, E., Hieta, R., Huntley, R., Legge, D., Liu, W.D., Luo, J., MacDougall, A., Mutowo, P., Nightingale, A., Orchard, S., Pichler, K., Poggioli, D., Pundir, S., Pureza, L., Qi, G.Y., Rosanoff, S., Sawford, T., Shypitsyna, A., Turner, E., Volynkin, V., Wardell, T., Watkins, X., Zellner, H., Corbett, M., Donnelly, M., Van Rensburg, P., Goujon, M., McWilliam, H., Lopez, R., Xenarios, I., Bougueleret, L., Bridge, A., Poux, S., Redaschi, N., Aimo, L., Auchincloss, A., Axelsen, K., Bansal, P., Baratin, D., Binz, P.A., Blatter, M.C., Boeckmann, B., Bolleman, J., Boutet, E., Breuza, L., Casal-Casas, C., de Castro, E., Cerutti, L., Coudert, E., Cuche, B., Doche, M., Dornevil, D., Duvaud, S., Estreicher, A., Famiglietti, L., Feuermann, M., Gasteiger, E., Gehant, S., Gerritsen, V., Gos, A., Gruaz-Gumowski, N., Hinz, U., Hulo, C., James, J., Jungo, F., Keller, G., Lara, V., Lemercier, P., Lew, J., Lieberherr, D., Lombardot, T., Martin, X., Masson, P., Morgat, A., Neto, T., Paesano, S., Pedruzzi, I., Pilbout, S., Pozzato, M., Pruess, M., Rivoire, C., Roechert, B., Schneider, M., Sigrist, C., Sonesson, K., Staehli, S., Stutz, A., Sundaram, S., Tognolli, M., Verbregue, L., Veuthey, A.L., Wu, C.H., Arighi, C.N., Arminski, L., Chen, C.M., Chen, Y.X., Garavelli, J.S., Huang, H.Z., Laiho, K., McGarvey, P., Natale, D.A., Suzek, B.E., Vinayaka, C.R., Wang, Q.H., Wang, Y.Q., Yeh, L.S., Yerramalla, M.S., Zhang, J.: Consortium U, activities at the Universal Protein Resource (UniProt). Nucleic Acids Res. 42(D1), D191–D198 (2014)Google Scholar

Copyright information

© American Society for Mass Spectrometry 2014

Authors and Affiliations

  1. 1.Department of BiochemistryDuke University Medical CenterDurhamUSA
  2. 2.Department of ChemistryDuke UniversityDurhamUSA

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