Transcription factors (suite)

From the technical point of view, MS-based identification of proteins after DNA-affinity capture represents a real challenge, in terms of sensitivity, as most transcriptional regulators are of low abundance, as well as in specificity as number of abundant proteins are unspecifically captured by the negatively charged DNA and/or by the chromatographic support (reviewed in Tacheny et al, J. Proteomics 2013). We have overcome these challenges by i) an efficient and original separation step of the protein/DNA complexes from the solid support, based on the reversible desthiobiotine-streptavidin interaction, ii) an adapted chromatographic separation of the complex peptide mixture and iii) a specific MS analysis focused on the identification of low abundant proteins. Remarkably, this assay can be applied to any DNA sequence, up to 300 bp long, without prior localisation of regulatory transcription factor binding sites.

We currently collaborate with several groups interested in using this method to decipher the proteins controlling their gene of interest.

Finally, we are currently developing a similar approach to identify the proteins binding to a selected RNA sequence