Identification of histone post-translational modifications and epigenetic complex members by mass spectrometry

AbstractIntroduction: The differentiation of stem cells constitutes a process poorly characterized at the epigenetic level. The contribution to the understanding of this process is extremely valuable in the advance of molecular-based therapies. After introducing the applicability of a new MS- based approach we here presented its potential in the investigation of changes in histone post-translational modifications during cell differentiation. The investigation of the protein epigenetic complexes is the other piece that completes the information about this process. The changes during differentiation of one of these complexes, the Polycomb group, were also attempted in this work.Methods: The sensitivity offered by mass spectrometry is extremely helpful in the identification of histone epigenetic marks. To increase the detection of low abundant species we presented here the combination of the accurate inclusion mass screening approach with the chemical derivatization of histones. After proving the robustness of this combined approach in the two different cell lines, NT2 and HEK293, we used to identify histone modifications during the cell differentiation of NT2 cells. The sensitivity of mass spectrometry was also helpful in the detection of new interactor of Polycomb members during the differentiation; the cell lines used were HMEC and HEK293.Results: The used of the combination of the two technologies presented here increased in some cases by two-fold the detection of histone marks. The application of this combined approach to the NT2 differentiation allowed the classification of 41 histone marks as constant, early or late expressed during this process; 9 of them were novel. The identification of a novel Polycomb interactor, the demethylase NO66, was also achieved.Discussion: The combined approach proved to identify an almost similar number of histone marks that more sophisticated methodologies. It also associated 9 new histone marks with not reported function to date with the differentiation of NT2 cells. Future work employing semisynthetic nucleosomes is proposed to investigate the role of these marks. The discovery of the interaction of NO66 with Polycomb led to further investigation that concluded in the publication of biological model of its role within the cell.