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 st ructures can lead to comprehension of their role in vivo and contribute to the discovery of structure-related gene regulation. This work was devoted t o establishing a reliable procedure to select proteins on the basis of thei r interaction with a nucleic acid probe chosen to fold into a given structu re. PD-electrophoresis and mass spectrometry were combined for protein iden tification. We applied this procedure to select and identify tripler-bindin g 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 o f spots was reproducibly revealed by South-western blotting. Spots of inter est were localised among a complex population of S-35- labelled proteins ac cording to their P-32-specific emission. Position of the same spots was ext rapolated on a preparative gel coloured with Coomassie blue, allowing excis ion and purification of the corresponding proteins. The material was subjec ted to mass spectrometry upon trypsin digestion and MALDI-TOF peptide finge rprinting was used for research in databases: five of them were identified and found to belong to the hnRNP family (K, L, A2/B1, El and I). The identi ties of several of them were confirmed by comparing western and South-weste rn blots on the same membrane using specific antibodies. The recognition sp ecificity of most of these proteins is large, according to previous reports and our own experiments. It includes pyrimidine-rich DNA sequences in diff erent contexts: single strand to a small extent, tripler and possibly other higher-order structures.