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Rapid identification of DNA-binding proteins by mass spectrometry

Abstract

We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The determined molecular masses are often sufficient for identification. If not, the proteins are subjected to mass spectrometric peptide mapping followed by database searches. Apart from protein identification, the protocol also yields information on posttranslational modifications. The protocol was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA.

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Figure 1: Identification of cyclic adenosine monophosphate receptor protein (CRP) in E. coli crude cell extract.
Figure 2: Identification of ectopically expressed rat RXRα in cell extracts from control yeast (A) or yeast expressing rat RXRα (B).
Figure 3: Identification of RXRα and PPARγ in 3T3-F442A crude nuclear extract.
Figure 4: Specific binding of HeLa cell nuclear proteins to the EFP1 element.
Figure 5: Identification of poly(ADP-ribose) polymerase (PARP) captured by the EFP1 probe.

References

  1. Fields, S. & Song, O. A novel genetic system to detect protein-protein interactions. Nature 340, 245– 246 (1989).

    CAS  Article  Google Scholar 

  2. Rebar, E.J. & Pabo, C.O. Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. Science 263, 671–673 (1994).

    CAS  Article  Google Scholar 

  3. Karas, M. & Hillenkamp, F. 1988.Laser desorption ionization of proteins with molecular masses exceeding 10,000 Daltons. Anal. Chem. 60, 2299–2301 .

    CAS  Article  Google Scholar 

  4. Gobom, J., Nordhoff, E., Ekman, R. & Roepstorff, P. Rapid micro-scale proteolysis of proteins for MALDI-MS peptide mapping using immobilized trypsin. Int. J. Mass Spectrom. Ion Proc. 169/170, 153–163 (1997).

    CAS  Article  Google Scholar 

  5. Henzel, W.J., Billeci, T.M., Stults, J.T. & Wong, S.C. Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc. Natl. Acad. Sci. USA 90, 5011–5015 (1993).

    CAS  Article  Google Scholar 

  6. Parkinson, G. et al. Structure of the CAP-DNA complex at 2.5 angstroms resolution: a complete picture of the protein-DNA interface. J. Mol. Biol. 260, 395–408 (1996).

    CAS  Article  Google Scholar 

  7. Kolb, A., Busby, S., Buc, H., Garges, S. & Adhya, S. Transcriptional regulation by cAMP and its receptor protein. Annu. Rev. Biochem. 62, 749– 795 (1993).

    CAS  Article  Google Scholar 

  8. Valentin-Hansen, P., Svenningsen, B.A., Munch-Pedersen, A. & Hammer-Jespersen, K. Regulation of the deo operon in Escherichia coli: the double negative control of the deo operon by the cytR and deoR repressors in a DNA directed in vitro system. Mol. Gen. Genet. 159, 191–202 (1978).

    CAS  Article  Google Scholar 

  9. Heyduk, T. & Lee, J.C. Escherichia coli cAMP receptor protein: evidence for three protein conformational states with different promoter binding affinities. Biochemistry 28, 6914 –6924 (1989).

    CAS  Article  Google Scholar 

  10. Allegretto, E.A. et al. Transactivation properties of retinoic acid and retinoid X receptors in mammalian cells and yeast. Correlation with hormone binding and effects of metabolism. J. Biol. Chem. 268, 26625 –26633 (1993).

    CAS  PubMed  Google Scholar 

  11. Henry, K., O'Brien, M.L., Clevenger, W., Jow, L. & Noonan, D.J. Peroxisome proliferator-activated receptor response specificities as defined in yeast and mammalian cell transcription assays. Toxicol. Appl. Pharmacol. 132, 317 –324 (1995).

    CAS  Article  Google Scholar 

  12. Lechler, A. & Kreutzer, R. . The phenylalanyl-tRNA synthetase specifically binds DNA. J. Mol. Biol. 278, 897–901 (1998).

    CAS  Article  Google Scholar 

  13. Um, S. et al. Retinoic acid receptors interact physically and functionally with the T:G mismatch-specific thymine-DNA glycosylase. J. Biol. Chem. 273, 20728–20736 (1998).

    CAS  Article  Google Scholar 

  14. Tontonoz, P., Spiegelman, B.M. & Hu, E. Stimulation of adipogenesis in fibroblasts by PPAR γ 2, a lipid-activated transcription factor. Cell 79, 1147–1156 (1994).

    CAS  Article  Google Scholar 

  15. Wakabayashi-Ito, N. & Nagata S. Characterization of the regulatory elements in the promoter of the human elongation factor-1 α gene. J. Biol. Chem. 269, 29831– 29837 (1994).

    CAS  PubMed  Google Scholar 

  16. Satoh, M. & Lindahl, T. Role of poly(ADP-ribose) formation in DNA repair. Nature 356, 356– 258 (1992).

    CAS  Article  Google Scholar 

  17. Tewari, M. et al. Yama/CPP32 β, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 81, 801–809 ( 1995).

    CAS  Article  Google Scholar 

  18. Meisterernst, M., Stelzer, G. & Roeder, R.G. . Poly(ADP-ribose) polymerase enhances activator-dependent transcription in vitro. Proc. Natl. Acad. Sci. USA 94, 2261–2265 (1997).

    CAS  Article  Google Scholar 

  19. Zhang, X., Gunasekera, A., Ebright, Y.W. & Ebright, R.H. Derivatives of CAP having no solvent-accessible cysteine residues, or having a unique solvent-accessible cysteine residue at amino acid 2 of the helix-turn-helix motif. J. Biomol. Struct. Dyn. 9, 463– 473 (1991).

    CAS  Article  Google Scholar 

  20. Dent, C.L. & Latchman, D.S. Transcription factors, a practical approach (Oxford University Press, Oxford; 1993).

    Google Scholar 

  21. Dignam, J.D., Lebovitz, R.M. & Roeder, R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic. Acids. Res. 11, 1475–1489 (1983).

    CAS  Article  Google Scholar 

  22. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 ( 1996).

    CAS  Article  Google Scholar 

  23. Gobom, J., Nordhoff, E., Mirgorodskaya, E., Ekman, R. & Roepstorff, P. Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry. J. Mass. Spectrom. 34, 105–116 (1999).

    CAS  Article  Google Scholar 

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Acknowledgements

This work was supported by grants from the Commission of the European Communities (TMR, ERBFMBICT950446) and the Danish Biotechnology Program.

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Correspondence to Eckhard Nordhoff.

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Nordhoff, E., Krogsdam, AM., Jørgensen, H. et al. Rapid identification of DNA-binding proteins by mass spectrometry. Nat Biotechnol 17, 884–888 (1999). https://doi.org/10.1038/12873

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