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.
Nordhoff
E
E
Department of Molecular Biology, Odense University, Odense, Denmark. nordhoff@mpimg-berlin-dahlem.mpg.de
Krogsdam
A M
AM
Jorgensen
H F
HF
Kallipolitis
B H
BH
Clark
B F
BF
Roepstorff
P
P
Kristiansen
K
K
D000818:Animals; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein / Q000302:isolation & purification; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008280:Magnetics; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods; D011065:Poly(ADP-ribose) Polymerases / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000302:isolation & purification / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000302:isolation & purification / Q000378:metabolism; D047488:Retinoid X Receptors; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D015342:DNA Probes; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D011065:Poly(ADP-ribose) Polymerases