A mass spectrometry-based method for detection of 5-formylcytosine binding proteins. (a) Schematic representation of the pull-down method. DNA oligonucleotides corresponding to the promoter regions of the Pax6 (280 bp) and Fgf15 (248 bp) genes were obtained by PCR with biotinylated primers and using dATP, dGTP. dTTP and either dCTP, dmCTP, dhmCTP or dfCTP. DNA was then incubated with Streptavidin-linked beads and with nuclear extract from mouse ES cells. Bound fraction was then eluted and analysed by mass spectrometry. (b) Western blot showing presence of UHRF1 in the protein fraction captured by methylated and hydroxymethylated probes (both Fgf15 and Pax6) compared to umodified DNA. (c, d) Venn diagrams and histograms showing distribution of significantly enriched proteins binding to differentially modified Fgf15 probe (CpG: 14; non-CpG: 69, %CpGs: 11.3%) and Pax6 probe (CpG: 8; non-CpG: 44; %CpG: 5.7%) with schematic representation of their genomic position.