The posttranslational modifications of core histones, such as the acetylation and methylation on lysine residues, play a critical role in a variety of gene activities such as transcription regulation, Deoxyribose Nucleic Acid (DNA) replication and damage repair, gene silencing and the regulation of cell developmental processes such as proliferation and differentiation. These modifications, as well as the responsible enzymes, can be related to the occurrence and development of many diseases such as leukemia, lupus and Huntington’s disease. A thorough investigation of these post-translational modification sites will shed light on the mechanism and function of histone modifications in these disorders.
Traditional methods for the investigation of histone post-translational modifications are primarily immunoassay techniques. These techniques are very sensitive, but they rely greatly on the availability of the site specific antibodies. The lack of a comprehensive set of antibodies has limited the studies of the novel modification sites. An additional complication with immunoassays arises when multiple modifications occur on the same histone. The simultaneous presence of other modifications may impact the specificity of antibodies directed at a specific individual modification.
In this study, mass spectrometry and its related techniques were applied for the investigation of histone post-translational modification. A model was first established to demonstrate that mass spectrometry and peptide mapping can be successfully applied to study the detailed localization of acetylation at specific lysine residues. Core histones were extracted from bovine thymus and identified by LC/MS. After the identification, these proteins were separated and digested using different enzymes. Direct peptide mapping was applied for these proteolytic digestion mixtures on a 7T FT ICR MS. The results showed more than 20 novel modification sites in addition to previously reported modification sites. Among them, the methylation of K59 in H4 was discovered to play a critical role in gene silencing.
High Performance Liquid Chromatography Mass Spectrometry (LC/MS) peptide mapping was also performed for the confirmation of these novel modification sites on histone H4 peptide fragments digested by trypsin and pepsin. The results supported the observation of novel modification sites observed with Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FT ICR MS). In addition, electron captured dissociation (ECD) was performed on H4 protein and its proteolytic digested fragments. Similar results were obtained as compared with peptide mapping.
As an application, LC/MS was used to determine the differential expression of the histone modifications in Acute Myeloid Leukemia (AML) and Chronic Lymphocytic Leukemia (CLL). The inhibition in different cell lines and leukemia patients caused by two Histone Deacetylase (HDAC) inhibitors were determined. Simultaneous observation was made for multiple modifications in all core histone.
Finally, another model was studied for quick identification and the semi-quantitative determination of histone acetylation. Deuterium-acetic anhydride (d6-acetic anhydride) was added to the partially acetylated protein/peptide for a D3-acetylation of the unmodified residues. D3-acetylation normalization was observed in peptide fragments containing lysine residues. The result is an easily identifiable isotope fingerprint that can be used as a “marker” for the investigation of the modification state of a particular fragment of the acetylated protein and peptide. Meanwhile, a simple comparison between the different D3-acetylated peak intensities will give us information regarding the composition of different modification isomers.