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Nucleic Acids Research, 2001, Vol. 29, No. 11 2427-2436
© 2001 Oxford University Press

Selection and identification of proteins bound to DNA triple-helical structures by combination of 2D-electrophoresis and MALDI-TOF mass spectrometry

F. Guillonneau, A. L. Guieysse, J. P. Le Caer1, J. Rossier1 and D. Praseuth*

Laboratoire de Biophysique, INSERM U201, CNRS UMR 8646, Muséum National d’Histoire Naturelle, 43 rue Cuvier, 75231 Paris Cedex 05, France and 1Laboratoire de Neurobiologie et diversité cellulaires, CNRS UMR 7637, E.S.P.C.I, 10 rue Vauquelin, 75231 Paris Cedex 05, France

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 35S-labelled proteins according to their 32P-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.

* To whom correspondence should be addressed. Tel: +33 1 40 79 37 08; Fax: +33 1 40 79 37 05; Email: praseuth{at}mnhn.fr


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