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Rapid identification of DNA-binding proteins by mass spectrometry


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.


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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).

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