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High throughput characterization of combinatorial histone codes.

ABSTRACT: We present a novel method utilizing "saltless" pH gradient weak cation exchange-hydrophilic interaction liquid chromatography directly coupled to electron transfer dissociation (ETD) mass spectrometry for the automated on-line high throughput characterization of hypermodified combinatorial histone codes. This technique, performed on a low resolution mass spectrometer, displays an improvement over existing methods with an approximately 100-fold reduction in sample requirements and analysis time. The scheme presented is capable of identifying all of the major combinatorial histone codes present in a sample in a 2-h analysis. The large N-terminal histone peptides are eluted by the pH and organic solvent weak cation exchange-hydrophilic interaction liquid chromatography gradient and directly introduced via nanoelectrospray ionization into a benchtop linear quadrupole ion trap mass spectrometer equipped with ETD. Each polypeptide is sequenced, and the modification sites are identified by ETD fragmentation. The isobaric trimethyl and acetyl modifications are resolved chromatographically and confidently distinguished by the synthesis of mass spectrometric and chromatographic information. We demonstrate the utility of the method by complete characterization of human histone H3.2 and histone H4 from butyrate-treated cells, but it is generally applicable to the analysis of highly modified peptides. We find this methodology very useful for chromatographic separation of isomeric species that cannot be separated well by any other chromatographic means, leading to less complicated tandem mass spectra. The improved separation and increased sensitivity generated novel information about much less abundant forms. In this method demonstration we report over 200 H3.2 forms and 70 H4 forms, including forms not yet detected in human cells, such as the remarkably highly modified histone H3.2 K4me3K9acK14acK18acK23acK27acK36me3. Such detail provided by our proteomics platform will be essential for determining how histone modifications occur and act in combination to propagate the histone code during transcriptional events and could greatly enable sequencing of the histone component of human epigenomes.

SUBMITTER: Young NL

PROVIDER: S-EPMC2758755 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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High throughput characterization of combinatorial histone codes.

Young Nicolas L NL DiMaggio Peter A PA Plazas-Mayorca Mariana D MD Baliban Richard C RC Floudas Christodoulos A CA Garcia Benjamin A BA

Molecular & cellular proteomics : MCP 20090804 10

We present a novel method utilizing "saltless" pH gradient weak cation exchange-hydrophilic interaction liquid chromatography directly coupled to electron transfer dissociation (ETD) mass spectrometry for the automated on-line high throughput characterization of hypermodified combinatorial histone codes. This technique, performed on a low resolution mass spectrometer, displays an improvement over existing methods with an approximately 100-fold reduction in sample requirements and analysis time. ...[more]

PMID: 19654425

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Project description:High throughput materials discovery using combinatorial polymer microarrays to screen for new biomaterials with new and improved function is established as a powerful strategy. Here we combine this screening approach with high throughput surface characterization (HT-SC) to identify surface structure-function relationships. We explore how this combination can help to identify surface chemical moieties that control protein adsorption and subsequent cellular response. The adhesion of human embryoid body (hEB) cells to a large number (496) of different acrylate polymers synthesized in a microarray format is screened using a high throughput procedure. To determine the role of the polymer surface properties on hEB cell adhesion, detailed HT-SC of these acrylate polymers is carried out using time of flight secondary ion mass spectrometry (ToF SIMS), X-ray photoelectron spectroscopy (XPS), pico litre drop sessile water contact angle (WCA) measurement and atomic force microscopy (AFM). A structure-function relationship is identified between the ToF SIMS analysis of the surface chemistry after a fibronectin (Fn) pre-conditioning step and the cell adhesion to each spot using the multivariate analysis technique partial least squares (PLS) regression. Secondary ions indicative of the adsorbed Fn correlate with increased cell adhesion whereas glycol and other functionalities from the polymers are identified that reduce cell adhesion. Furthermore, a strong relationship between the ToF SIMS spectra of bare polymers and the cell adhesion to each spot is identified using PLS regression. This identifies a role for both the surface chemistry of the bare polymer and the pre-adsorbed Fn, as-represented in the ToF SIMS spectra, in controlling cellular adhesion. In contrast, no relationship is found between cell adhesion and wettability, surface roughness, elemental or functional surface composition. The correlation between ToF SIMS data of the surfaces and the cell adhesion demonstrates the ability to identify surface moieties that control protein adsorption and subsequent cell adhesion using ToF SIMS and multivariate analysis.

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High throughput characterization of combinatorial histone codes.

Publications

High throughput characterization of combinatorial histone codes.

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