Characterization of Histone H2A and H2B Variants and Their Post-translational Modifications by Mass Spectrometry

Abstract

The nucleosome, the fundamental structural unit of chromatin, contains an octamer of core histones H3, H4, H2A, and H2B. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function, analysis of histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2A and H2B variants derived from Jurkat cells. A combination of mass spectrometric techniques, HPLC separations, and enzymatic digestions using endoproteinase Glu-C, endoproteinase Arg-C, and trypsin were used to identify histone H2A and H2B subtypes and their modifications. We identified nine histone H2A and 11 histone H2B subtypes, among them proteins that only had been postulated at the gene level. The two main H2A variants, H2AO and H2AC, as well as H2AL were either acetylated at Lys-5 or phosphorylated at Ser-1. For the replacement histone H2AZ, acetylation at Lys-4 and Lys-7 was found. Within the eukaryotic cell nucleus the genetic information is organized in a highly conserved structural polymer, termed chromatin, that supports and controls crucial functions of the genome. The fundamental unit of eukaryotic chromatin, the nucleosome, consists of 146 base pairs of genomic DNA wrapped around an octamer of the core histone proteins H2A, H2B, H3, and H4. The amino-terminal tails of each of the four core histones are subject to several types of covalent modifications, including acetylation, methylation, and phosphorylation. These modifications affect lysines (acetylation, mono-, di-, and trimethylation), serines and threonines (phosphoryla-

Liquid chromatography mass spectrometry profiling of histones

“…H2A and H2B have been reported to possess acetylation (H2A: K5, K9 and H2B: K5, K12, K15, K20), phosphorylation (H2A: S1 and H2B: S10), monomethylation (H2A: K15 and H2B: K5, K43) and ubiquitination (H2A: K119 and H2B: K120/K123) [1,14,16,[36][37][38][39][40]. Thus the unambiguous assignment of the species in these profiles is complicated by possible presence of these modifications along with the variants.…”

“…This analysis would not be possible by direct LC-MS/MS. The sequence variation within the human H2A variants are listed in Table 2 [63]. Specific peptide sequences were used to determine the presence of a unique form.…”

“…Such profiles of histones can be used as an assay to determine changes in chromatin that are a function of disease or treatment. Several investigators have dedicated their research efforts to the development of LC-MS methods for the analysis of histones [14][15][16]. These studies have primarily focused on the characterization of core histones.…”

“…For example, Zhang and Tang have applied micorbore C4 RP LC-MS to analyze the core histones from the chicken blood [17]. Bonenfant et al applied C18 RP LC-MS to the study of histones derived from Jurkat cells [16]. Hydrophilic-interaction liquid chromatography (HILIC) coupling with off-line mass spectrometry has been successful in separating and characterizing different histone isoforms [18,19].…”

Comparing and Combining Capillary Electrophoresis Electrospray Ionization Mass Spectrometry and Nano–Liquid Chromatography Electrospray Ionization Mass Spectrometry for the Characterization of Post-translationally Modified Histones

“…Over the past several years, considerable efforts have been expended to develop methods to identify the specific sites of histone modifications. Mass spectrometry (MS) coupled to liquid chromatography (LC) is the dominant technique for their characterization (7)(8)(9)(10)(11)(12)(13)(14). However, because histone proteins contain up to nearly 35% basic amino acids, the analysis of histone peptides is still problematic, as digestion with many commonly used enzymes (e.g.…”