This dissertation describes both the biological and technical aspects of one specific epigenetic template: histones and their modifications.

In this dissertation, we profile the histone epigenome of human embryonic stem cells (hESCs) during conversion from the primed to the naive state using an untargeted mass spectrometry (MS)-based approach. In total, 23 histone post-translational modifications (hPTMs) changed significantly over time. H3K27me3 was the most prominently increasing marker hPTM in naive hESCs. This is in line with what was recently described in mouse, prompting us to compare all the shared hPTM fold changes between mouse and human, revealing a set of conserved hPTM markers for the naive state. Principally, we present the first roadmap of the changing human histone epigenome during the conversion of hESCs from the primed to the naive state. This further revealed similarities with mouse, which hint at a conserved mammalian epigenetic signature of the ground state of pluripotency.

Sequential window acquisition of all theoretical fragment ion spectra (SWATH)-MS has great potential for large-scale analysis of hPTMs and their combinatorial patterns, as it allows for untargeted accurate identification and quantification of hPTMs. In this dissertation, we present a complete SWATH workflow specifically adapted for the untargeted study of histones (hSWATH). More specifically, the workflow was optimized to ensure a maximal coverage of the histone code and a high specificity for isobaric and co-eluting peptides. We assessed the validity of the workflow on a technical dataset of a time-lapse deacetylation of a commercial histone extract using HDAC1, which contains a ground truth, i.e. acetylated substrate peptides reduce in intensity. Finally we successfully apply this workflow in a biological setting and subsequently investigate the differential response to HDAC inhibition in different breast cancer cell lines.