Post-translational modifications (PTM’s) on histones plays a crucial role in determining the immune cell's fate in terms of differentiation, development, and disease. Mass spectrometry has emerged as a powerful tool for global histone PTM studies. Analysis of histone PTM’s is very complex owing to the enormous number and types of isobaric histone peptides. Histone has four forms (H2A, H2B, H3, and H4) attached with a linker H1 and multiple isoforms.
We used a “bottom-up” MS-based proteomics strategy to identify histone PTMs and to determine their abundance in CD8+ T cells of the mouse. Briefly, histones proteins were subjected to chemical derivatization by Propionic anhydride to generate peptides suitable for MS analysis. The MS was operated in DDA mode and data analysis was carried out with Epiprofile 2.1 basic and Proteome discoverer 2.3 (PD) against the UniProt database. Epiprofile is a MATLAB-based software specially developed for Histone PTM’s analysis using Hela cells. EpiProfile was designed to extract ions for a defined set of histone peptides (5 Histones, 24 isoforms, 70 peptides, and 213 modified/unmodified forms) by determining the RT for non-isobaric peptides by the MS1 and discriminate isobaric peptides using unique fragment ions in the MS2 spectra. We found that the Epiprofile worked well however comparison of the Epiprofile results with our PD results revealed that only 31 peptides were found in common between the two analysis tools. The limited library of peptides in Epiprofiler limiting its utility. Using PD we found 5 Histones, 28 isoforms, and 700 modified/unmodified forms of 160 histone peptides. The limited overlap between two tools is a direct example of the complexity of histone modifications and the differences between the Hela cells used for the development of Epiprofile and the CD8+ T cells used in our own study. This work will now form the basis of our future work examining histone modifications and epigenetic regulation of CD8+ T cells.