Decoding the histone code: Analysis of histone post-translational modifications by mass spectrometry

Histone proteins are the fundamental building blocks of eukaryotic chromatin. A diverse array of post-translational modifications (PTMs), such as acetylation, phosphorylation and methylation are found on the N-terminal tails of these proteins. The modifications are hypothesized to inscribe a code that regulates all chromatin-mediated processes. Here a derivatization strategy was employed that allowed for the comprehensive and comparative analysis of histone proteins using nanoflow RP-HPLC mu-electrospray ionization mass spectrometry (mu-ESI-MS). The epsilon-amino groups of lysine residues were converted to propionyl amides. A second derivatization strategy was employed that introduced a stable isotope tag on every carboxy moiety of generated tryptic histone peptides and enabled us to compare the pattern of modifications between two different samples. A large number of peptides were identified carrying single or multiple previously known and novel modifications. The findings led to the refinement of the previously published histone code hypothesis and gave birth to a new hypothesis---binary switches and modification cassettes.; In a different approach, a combination of gas-phase ion/ion chemistry and tandem mass spectrometry (MS/MS) has been applied for the analysis of underivatized larger histone peptides and intact proteins. Singly charged fluoranthene anions transferred an electron to multiply protonated peptides in a quadrupole linear ion trap (QLT) and induced fragmentation of the peptide backbone along pathways that were analogous to those observed in electron capture dissociation. A second ion/ion reaction utilizing deprotonated benzoic acid was employed to simplify mixtures of highly charged product ions into predominantly singly charged ions. Key biological N-terminal peptides of histone H4 (1--23) and H2B (1--24) and their modification patterns were characterized for the first time with near-complete sequence coverage (>90%). The sequential ion/ion reactions were further applied to larger historic peptides of histone H3 (50 residues), and to entire histone proteins and non-histone proteins of up to 66 kDa. Finally, this technique was applied to an unknown intact histone sample, and identification of an as yet unknown H2A family member was achieved.; The developed methodologies are applicable to histone samples from various sources and can be used to map post-translational modifications or to study changes in modification patterns.