Histone H3 tail interactome in MPC11 murine myeloma cells

We have used peptide capture in conjunction with super-SILAC quantification to carry out an unbiased high-throughput analysis of the composition of protein complexes that bind to histone H3K9/S10 and H3K27/S28 methyl-phospho modifications. We compared binding profiles of nuclear proteins from MPC11 myeloma cells to peptides carrying the H3K9me3, H3K27me3, H3S10ph, H3S28ph and the double H3K9me3/S10ph and H3K27me3/S28ph modifications. The analysis was carried out using peptides corresponding to amino acids 1-20 and 18-38 and the unmodified peptides were also analysed to assess binding to the unmodified histone tail. In order to ensure that the proteins present in any of the "heavy" capture assay samples have corresponding peptides in the common "light" internal reference, we adapted the super-SILAC approach using a reference sample that was obtained by mixing the 8 pulldowns and the negative beads-only control. The method allows us to accurately quantify binding and directly compare levels of binding to peptides that carry a number of different methyl-phospho combinations. This makes it possible for use indeatiled bioinformatic analyses.