Selection and identification of proteins bound to DNA triple-helical structures by combination of 2D-electrophoresis and MALDI-TOF mass spectrometry.
Identification of proteins binding specifically to peculiar nucleic acid structures can lead to comprehension of their role in vivo and contribute to the discovery of structure-related gene regulation. This work was devoted to establishing a reliable procedure to select proteins on the basis of their interaction with a nucleic acid probe chosen to fold into a given structure. 2D-electrophoresis and mass spectrometry were combined for protein identification. We applied this procedure to select and identify triplex-binding activities in HeLa nuclear extracts. To achieve this, we used a panel of deoxyribonucleic probes adopting intramolecular triple-helices, varying in their primary sequence, structure or triple-helix motif. A limited number of spots was reproducibly revealed by South-western blotting. Spots of interest were localised among a complex population of (35)S-labelled proteins according to their (32)P-specific emission. Position of the same spots was extrapolated on a preparative gel coloured with Coomassie blue, allowing excision and purification of the corresponding proteins. The material was subjected to mass spectrometry upon trypsin digestion and MALDI-TOF peptide fingerprinting was used for research in databases: five of them were identified and found to belong to the hnRNP family (K, L, A2/B1, E1 and I). The identities of several of them were confirmed by comparing western and South-western blots on the same membrane using specific antibodies. The recognition specificity of most of these proteins is large, according to previous reports and our own experiments. It includes pyrimidine-rich DNA sequences in different contexts: single strand to a small extent, triplex and possibly other higher-order structures.
Guillonneau
F
F
Laboratoire de Biophysique, INSERM U201, CNRS UMR 8646, Muséum National d'Histoire Naturelle, 43 rue Cuvier, 75231 Paris Cedex 05, France.
Guieysse
A L
AL
Le Caer
J P
JP
Rossier
J
J
Praseuth
D
D
D001483:Base Sequence; D015153:Blotting, Western; D002457:Cell Extracts / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012261:Ribonucleoproteins / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002457:Cell Extracts; D004268:DNA-Binding Proteins; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D012261:Ribonucleoproteins; C439768:hnRNP A2; D004247:DNA