Expanding the mass spectrometry toolkit for interrogating chromatin proteomics

Chromatin is comprised of DNA and a vast network of proteins, which help structure and regulate a cell’s genetic material through processes including cell differentiation, regulation of genes, and DNA repair. At the core of chromatin structure lays a nucleosome, consisting of DNA wrapped around an octameric protein complex made of histones. Histones can undergo post translational modifications (PTMs) which govern protein-protein interactions (PPIs) and ultimately control gene activation and suppression. Histone PTMs can already be quantitated using existing well defined methods such as liquid chromatography mass spectrometry (LC-MS). However with the increasing popularity of techniques such as chromatin immunoprecipitation aimed at identifying and understanding the role of PTMs under very specific circumstances, ever smaller amounts of histones are being produced and push heavily on the limits of LC-MS detection. We were able to reduce the number of cells required for a typical histone PTM LC-MS analysis from 10^6 cells to 10^5 cells and permitting technical replicates at this level. Chemical cross-linking mass spectrometry is another useful tool in characterising PPIs whilst simultaneously providing limited structural information, even from native cellular environments and cell lysates making it highly promising for chromatin. Much development has been made in this technology, however data analysis for this technique can still be difficult and laborious. We proposed to address this by simplifying the complexity of the data by altering the functional chemistry of the cross-linker, with limited levels success. Finally, some molecules have great therapeutic potential in addressing erroneous chromatin regulations that are implicated in a number of cancers. In order for more efficient therapeutics to be developed, it is important to identify how and where they bind to proteins. We were able to address this issue for a ligand-protein pair, for a protein known to be implicated in cancer and modify histones.Open Acces