Determination of acetylation and methylation sites of histones by mass spectrometry

This dissertation describes the development of a rapid method for determining the acetylation and methylation sites of core histories (H2B, H2A, H3 and H4) that gives the relative abundance of the acetylation isoforms by a method superior to the traditional micro-chemical sequencing method. The method created in this dissertation emphasizes speed, sensitivity and general applicability to all sorts of histone acetylation (methylation) scenarios. In this study, mass spectrometry method was first used to identify histone acetylation and methylation sites. Although there are 15 possible acetylation sites, only four acetylated peptide sequences were observed. The tetra-acetylated form is acetylated at lysines 5, 8, 12 and 16, the tri-acetylated form is mostly acetylated at lysines 8, 12 and 16, and the diacetylated form is acetylated at lysine 12 and 16. The only significant amount of mono-acetylated form is modified at lysine 16. These results are consistent with the hypothesis of a “zip” model whereby acetylation of histone H4 proceeds in the direction from Lys16 to Lys5 and deacetylation goes in the reverse direction. This acetylation pattern is conserved in mammal species including HeLa cells, colon carcinoma cells and chicken erythrocyte. Core histones from chicken erythrocyte were isolated by the acid precipitation method. LC/MS simultaneously identified each subclass of histories and its potential acetylated or methylated isoforms. Acetylation and methylation sites of chicken core histories were identified. Histone H4 is acetylated at lysine residues 16, 12, 8 and 5 in the direction from lysine 16 to 5 as observed in butyrate treated HeLa cells. Lysine 20 in all isoforms of histone H4 is predominately di-methylated. About 17% of H2A is acetylated with a roughly even distribution of mono-acetylated at lysine 9 and di-acetylated at lysines 5 and 9. Lysine residues 18 and 23 in H3 are acetylated in the direction from lysine 23 to lysine 18 based on the observation that if lysine 18 is acetylated, lysine 23 also acetylated. Lysine 14 was determined to be partially acetylated (two thirds), partially methylated (one third) by high accuracy mass measurement. Lysine 4 of H3 is un- and mono-methylated. Lysine residues 9, 27 and 36 in H3 are un-, mono-, di- and trimethylated while lysine 79, a newly found methylation site for H3, is un, mono- and dimethylated. H2B is partially methylated (about 50%) at lysine 30 with a rough distribution of 1 : 0.4 : 0.3 : 0.2 for un-, mono-, di- and tri-methylated forms. (Abstract shortened by UMI.