Characterization of Tetrahymena Histone H2B Variants and Posttranslational Populations by Electron Capture Dissociation (ECD) Fourier Transform Ion Cyclotron Mass Spectrometry (FT-ICR MS)

Medzihradszky, Katalin F.; Zhang, X.; Chalkley, Robert J.; Guan, Shenheng; McFarland, Melinda A.; Chalmers, Michael J.; Marshall, Alan G.; Diaz, Robert L.; Allis, C. David; Burlingame, A. L.

doi:10.1074/mcp.m400041-mcp200

Cited by 89 publications

(88 citation statements)

References 46 publications

(110 reference statements)

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“…Imagine the code as a complete sentence, antibodies can identify letters or sometimes words, but these words and letters lack context. Mass spectrometry-based (MS) sequencing methods, however, are rapidly evolving, have high sensitivity, and can identify and quantitate PTM patterns without a priori knowledge (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Here, we have developed and applied a method for the discovery and quantification of histone H4 combinatorial codes that is based on chromatography and recently developed MS technology.…”

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confidence: 99%

Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells

Phanstiel

Brumbaugh²,

Berggren³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

162

175

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Epigenetic regulation through chromatin is thought to play a critical role in the establishment and maintenance of pluripotency. Traditionally, antibody-based technologies were used to probe for specific posttranslational modifications (PTMs) present on histone tails, but these methods do not generally reveal the presence of multiple modifications on a single-histone tail (combinatorial codes). Here, we describe technology for the discovery and quantification of histone combinatorial codes that is based on chromatography and mass spectrometry. We applied this methodology to decipher 74 discrete combinatorial codes on the tail of histone H4 from human embryonic stem (ES) cells. Finally, we quantified the abundances of these codes as human ES cells undergo differentiation to reveal striking changes in methylation and acetylation patterns. For example, H4R3 methylation was observed only in the presence of H4K20 dimethylation; such context-specific patterning exemplifies the power of this technique.electron transfer dissociation ͉ epigenetics ͉ posttranslational modification ͉ histone code ͉ pluripotency P luripotency-the ability to differentiate into any specialized lineage-is the hallmark of embryonic stem (ES) cells and the basis for their experimental and therapeutic potential. The precise molecular mechanisms that define pluripotency remain elusive; however, a number of recent works suggest a central role for epigenetic regulation through chromatin (1-6). Either by recruiting or shielding certain factors, modifications on histone proteins modulate a gene's local environment and thereby regulate expression (7-12). Concerted changes in histone modification states occur during differentiation (13). For example, high levels of histone H3 and H4 acetylation are characteristic of pluripotent cells in mice and the abundance of these marks decreases during differentiation (14, 15). Methylation of R2, R17, and R26 of histone H3 by CARM1 also correlates with cell fate and potency (6). Cells with higher levels of methylation, at these residues, were enriched in the embryonic part of the blastocyst. Next, specific patterns of histone H3K4me3 and H3K27me3 are observed at promoter regions of genes that are regulated during differentiation (1, 2). Finally, demethylation of H3K27me3 is required for activation of certain HOX genes essential for proper development (16,17). The demethylase responsible interacts directly with MLL 2/3 complexes, which methylate histone H3K4 (18). Taken together these experiments have shed new light on the power of epigenetic regulation within ES cells; however, the precise details and role(s) of such combinatorial PTM patterns remain largely unknown.Technological limitations in our ability to discover and quantify combinatorial histone PTMs has, and continues to, present a major obstacle. Most of our knowledge of epigenetics has been derived by antibody-based approaches. Antibodies require a priori knowledge of individual modifications, are subject to epitope occlusion, and have difficulty distingu...

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mentioning

confidence: 99%

Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells

Phanstiel

Brumbaugh²,

Berggren³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

162

175

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“…This ''top-down'' analysis (21) has been successfully used to determine histone PTMs. Using ECD, Kelleher and coworkers (22)(23)(24)(25) characterized PTMs on all four human core histones, Burlingame and colleagues (26) characterized Tetrahymena H2B variants, and Zhang and Freitas (27) characterized histone H4 from calf thymus. Recently, an ion/ion analogue of ECD, termed electron transfer dissociation (ETD) (28), was developed.…”

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confidence: 99%

Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini

Taverna

Ueberheide

Liu

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

174

191

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Individual posttranslational modifications (PTMs) on histones have well established roles in certain biological processes, notably transcriptional programming. Recent genomewide studies describe patterns of covalent modifications, such as H3 methylation and acetylation at promoters of specific target genes, or ''bivalent domains,'' in stem cells, suggestive of a possible combinatorial interplay between PTMs on the same histone. However, detection of long-range PTM associations is often problematic in antibody-based or traditional mass spectrometric-based analyses. Here, histone H3 from a ciliate model was analyzed as an enriched source of transcriptionally active chromatin. Using a recently developed mass spectrometric approach, combinatorial modification states on single, long N-terminal H3 fragments (residues 1-50) were determined. The entire modification status of intact N termini was obtained and indicated correlations between K4 methylation and H3 acetylation. In addition, K4 and K27 methylation were identified concurrently on one H3 species. This methodology is applicable to other histones and larger polypeptides and will likely be a valuable tool in understanding the roles of combinatorial patterns of PTMs.bivalent domain ͉ electron transfer dissociation ͉ mass spectrometry ͉ posttranslational modifications ͉ Tetrahymena

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“…Several ion manipulation techniques and use of a large, movable dispenser cathode reduce the possibility that misalignment of the ion and electron beams limits ECD efficiency. [12], and histones [13][14][15]. Despite its proven analytical utility, ECD suffers from limited conversion efficiency of precursor to product ions.…”

mentioning

confidence: 99%

“…(J Am Soc Mass Spectrom 2005, 16, 1060 -1066) © 2005 American Society for Mass Spectrometry E lectron capture dissociation (ECD) [1,2] is a relatively new MS/MS technique that has become popular because it provides better peptide and protein sequence coverage compared with other dissociation methods, and retains labile post-translational modifications (e.g., glycosylation [3,4] and phosphorylation [5][6][7][8]. Recent applications also include fatty acids [9], antibiotic/protein complexes [10], ubiquitination [11], oligonucleotides [12], and histones [13][14][15]. Despite its proven analytical utility, ECD suffers from limited conversion efficiency of precursor to product ions.…”

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confidence: 99%

Evaluation and optimization of electron capture dissociation efficiency in fourier transform ion cyclotron resonance mass spectrometry

McFarland

Chalmers

Quinn

et al. 2005

J. Am. Soc. Mass Spectrom.

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Electron capture dissociation (ECD) efficiency has typically been lower than for other dissociation techniques. Here we characterize experimental factors that limit ECD and seek to improve its efficiency. Efficiency of precursor to product ion conversion was measured for a range of peptide (ϳ15% efficiency) and protein (ϳ33% efficiency) ions of differing sizes and charge states. Conversion of precursor ions to products depends on electron irradiation period and maximizes at ϳ5-30 ms. The optimal irradiation period scales inversely with charge state. We demonstrate that reflection of electrons through the ICR cell is more efficient and robust than a single pass, because electrons can cool to the optimal energy for capture, which allows for a wide range of initial electron energy. Further, efficient ECD with reflected electrons requires only a short (ϳ500 s) irradiation period followed by an appropriate delay for cooling and interaction. Reflection of the electron beam results in electrons trapped in or near the ICR cell and thus requires a brief (ϳ50 s) purge for successful mass spectral acquisition. Further electron irradiation of refractory precursor ions did not result in further dissociation. Possibly the ion cloud and electron beam are misaligned radially, or the electron beam diameter may be smaller than that of the ion cloud such that remaining precursor ions do not overlap with the electron beam. Several ion manipulation techniques and use of a large, movable dispenser cathode reduce the possibility that misalignment of the ion and electron beams limits ECD efficiency. [12], and histones [13][14][15]. Despite its proven analytical utility, ECD suffers from limited conversion efficiency of precursor to product ions. The dispenser cathode electron source has decreased the irradiation period and increased the reproducibility of ECD [16], but has not significantly improved the efficiency [17]. Factors such as charge neutralization and larger number of observed fragmentation pathways make ECD difficult to apply to large precursor ions of low abundance. In addition, it can be quite difficult to establish and maintain optimized ECD operating conditions. The emergence of FT-ICR instruments in biological mass spectrometry makes the robustness and efficiency of ECD even more critical.In this work, we systematically characterize experimental factors that limit ECD and seek to improve its efficiency. Research is designed to investigate all aspects of the ECD experiment, including duration of electron irradiation, electron energy and flux, multiple reflection of the electron beam versus a single pass through the ICR cell, ejection of trapped electrons from the ICR cell following the ECD event, and spatial overlap of the electron beam with the trapped ions. We report our conditions for optimized ECD and discuss their physical rationale and general implications.

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Characterization of Tetrahymena Histone H2B Variants and Posttranslational Populations by Electron Capture Dissociation (ECD) Fourier Transform Ion Cyclotron Mass Spectrometry (FT-ICR MS)

Cited by 89 publications

References 46 publications

Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells

Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells

Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini

Evaluation and optimization of electron capture dissociation efficiency in fourier transform ion cyclotron resonance mass spectrometry

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