Combinatorial Modification of Human Histone H4 Quantitated by Two-dimensional Liquid Chromatography Coupled with Top Down Mass Spectrometry

Abstract

Quantitative proteomics has focused heavily on correlating protein abundances, ratios, and dynamics by developing methods that are protein expression-centric (e.g. isotope coded affinity tag, isobaric tag for relative and absolute quantification, etc.). These methods effectively detect changes in protein abundance but fail to provide a comprehensive perspective of the diversity of proteins such as histones, which are regulated by post-translational modifications. Here, we report the characterization of modified forms of HeLa cell histone H4 with a dynamic range >10 4 using a strictly Top Down mass spectrometric approach coupled with two dimensions of liquid chromatography. This enhanced dynamic range enabled the precise characterization and quantitation of 42 forms uniquely modified by combinations of methylation and acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quantitative analyses revealed distinct trends in acetylation site occupancy depending on Lys-20 methylation state. Because both modifications are dynamically regulated through the cell cycle, we simultaneously investigated acetylation and methylation kinetics through three cell cycle phases and used these data to statistically assess the robustness of our quantitative analysis. This work represents the most comprehensive analysis of histone H4 forms present in human cells reported to date.Histones are a class of proteins around which DNA is wrapped and packaged inside a eukaryotic nucleus. Two molecules of each core histone H2A, H2B, H3, and H4 together with ϳ146 bp of DNA form the fundamental unit of chromatin called the nucleosome. These proteins are heavily modified, with combinations of these enzymatic modifications thought to form a "histone code" orchestrating epigenetic processes such as long-term gene silencing and gene activation (1), higher level chromatin packaging (2), and DNA repair mechanisms (3). All of these activities change with relation to the cell cycle, a sequence of events during which a cell commits to DNA replication (G 1 ), replicates its DNA (S), prepares for mitosis (G 2 ), and undergoes cell division (M) (4). Histone synthesis and deposition are largely coupled to DNA replication during S phase (5). As a cell doubles its nuclear DNA, there is a concomitant doubling of the content of histones and nucleosomes. Even though antibodies have been used to track single modifications, the fate of preexisting histone modifications and the acquisition of new histone modifications during the cell cycle is not well understood because this approach is unable to distinguish previously modified forms from newly modified ones (6 -8). However, an epigenetic mechanism presumably exists to faithfully transmit patterns of histone modification and chromatin structure to ensure normal cellular function over successive generations (9).Dynamic changes in the PTMs 4 affecting the N-terminal tails of the core histones, which comprise ϳ25-30% of their individual...

A preferred AMPK phosphorylation site adjacent to the inhibitory loop of cardiac and skeletal troponin I

“…For quantification of cTnI phosphospecies, MS signal intensity values (the top five most abundant isotopomer peak heights) were integrated to calculate relative ratios. [31][32][33]62,63 Based on signal-to-noise ratios, the relative detection sensitivity was estimated to be about 1-3% of highest peak intensity for MS spectra and about 1-5% for ECD spectra.…”

Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state

“…Biomolecular mass spectrometry (MS) (21) is the only technique that can universally provide information about protein posttranslational modifications (PTMs) without a priori knowledge (22)(23)(24)(25)(26). In contrast to the conventional ''bottom-up'' MS approach where proteins of interest are digested with an enzyme before MS analysis providing only partial coverage of the protein sequence with loss of connectivity between modified peptides from disparate regions of the protein (23,27,28), a ''top-down'' MS approach is extremely attractive for characterization of complex PTMs in proteins of 10 to 200 kDa (22,25,(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39).…”

Chemical and biochemical approaches in the study of histone methylation and demethylation

“…117,155,[176][177][178] A great challenge in epigenetics research is to elucidate the biochemical effects of specific histone modifications on cell growth and differentiation. Histones isolated from mammalian cells possess complex and heterogeneous modification patterns, 151,163,179 which makes it technically difficult to investigate the contribution from individual modification sites. For example, methyllysine residues are hypothesized to mediate interactions between nucleosomal histones and macromolecular complexes that regulate DNA transcription, replication, and repair.…”

“…Recently Pesavento and colleagues applied top-down MS and hydrophilicinteraction liquid chromatography for the quantification of histone H4 combinatorial codes. 150,163 By combining efficient separation of intact protein, high accuracy FT-MS measurement and ECD fragmentation, enhanced dynamic range (>10 4 ) has been achieved that allows the precise characterization and quantification of 42 forms uniquely modified by methylation and acetylation. The dynamic changes of some low abundant methylation forms including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms) were also determined.…”