AbstractsChemistry

Characterization of histone post-translational modification using reversed-phase high performance liquid chromatography and fourier transform ion cyclotron resonance mass spectrometry

by Liwen Zhang




Institution: The Ohio State University
Department: Chemistry
Degree: PhD
Year: 2003
Keywords: Chemistry, Analytical; Histone Post-translational Modification; Peptide Mapping; Fourier Transform Ion Cyclotron Resonance Mass Spectrometry; High Performance Liquid Chromatography Mass Spectrometry; Tandem Mass Spectrometry
Record ID: 1741447
Full text PDF: http://rave.ohiolink.edu/etdc/view?acc_num=osu1054660495


Abstract

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…