This research was originally published in Mol Cell Proteomics. Soldi M. and Bonaldi T. The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components MCP. 2013; 12: 64-80. &#169; the American Society for Biochemistry and Molecular Biology. We thank Roberta Noberini (Italian Institute of Technology and IEO, Italy) for critical reading of the manuscript. TB work is supported by grants from the Giovanni Armenise-Harvard Foundation Career Development Program, the Italian Association for Cancer Research and the Italian Ministry of Health. MS work was supported by a FIRC fellowship.

<ol>
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No conflicts of interest declared.

We have recently described ChroP, a quantitative strategy for the large-scale characterization of the protein components of chromatin. ChroP combines two complementary approaches used in the epigenetic field, ChIP and MS, profiting from their strengths and overcoming their respective limitations. ChIP coupled to deep sequencing (ChIP-Seq) allows the genome-wide mapping of histone modifications at the resolution of few nucleosomes35. Although advantageous for their sensitivity, antibody-based assays are limited...

Chromatin is a highly dynamic nucleoprotein complex that is involved as primary template for all DNA-mediated processes. The nucleosome is the basic repeated unit of chromatin and consists of a proteinaceous octameric core containing two molecules of each canonical histone H2A, H2B, H3 and H4, around which 147 bp of DNA are wrapped1,2. All core histones are structured as a globular domain and a flexible N-terminal &#8220;tail&#8221; that protrudes outside the nucleosome. One of the major mechanisms for regulating chromatin structure and dynamics is based on covalent post-translational modifications (PTMs), which mainly occur on the N-termini of histones3,4. Histone modifications can function either by altering the higher order chromatin structure, by changing contacts between histones-DNA or between nucleosomes, and thus controlling the accessibility of DNA-binding proteins (cis mechanisms), or by acting as docking sites for regulatory proteins, either as single units, or embedded in multimeric complexes. Such regulating proteins can exert their function in different ways: by modulating directly gene expression (i.e. TAF proteins), or by altering the nucleosome positioning (i.e. chromatin remodeling complexes) or by modifying other histone residues (i.e. proteins with methyl-transferase or acetyl-transferase activity) (trans mechanisms)5. The observation that distinct PTM patterns cluster at specific chromatin loci led to the elaboration of the hypothesis that different modifications at distinct sites may synergize to generate a molecular code mediating the functional state of the underlying DNA. The &#34;histone code hypothesis&#34; has gained large consensus in the years but its experimental verification has been held back by technical limitations6,7.
Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to map histone modification patterns and to characterize chromatin-binding proteins8. MS detects a modification as a specific &#916;mass between the experimental and theoretical mass of a peptide. At the level of individual histones, MS provides an unbiased and comprehensive method to map PTMs, allowing the detection of new modifications and revealing interplays among them9-14.
In recent years, a number of strategies have been developed to dissect the proteomic composition of chromatin, including the characterization of intact mitotic chromosomes15, the identification of soluble hPTM-binding proteins16-18 and the isolation and analysis of specific chromatin regions (i.e. telomeres)19,20. However, the investigation of the locus-specific synergies between histone PTMs, variants, and chromatin-associated proteins is still incomplete. Here, we describe a new approach, named ChroP (Chromatin Proteomics)21, that we have developed to efficiently characterize functionally distinct chromatin domains. This approach adapts chromatin immunoprecipitation (ChIP), a well-established protocol used in epigenetic research, for the efficient MS-based proteomic analysis of the enriched sample. We have developed two different protocols, depending on the type of chromatin used as input and the question addressed by MS; in particular: 1) ChIP of unfixed native chromatin digested with MNase is employed to purify mono-nucleosomes and to dissect the co-associating hPTMs (N-ChroP); 2) ChIP of crosslinked chromatin fragmented by sonication is used in combination with a SILAC-based interactomics strategy to characterize all co-enriching chromatin binding proteins (X-ChroP). We illustrate here the combination of N- and X-ChroP for enriching and studying heterochromatin, using H3K9me3 as bait for the immunoprecipitation steps. The use of ChroP can be extended to study either distinct regions on chromatin, or changes in the chromatin composition within the same region during the transition to a different functional state, thus paving the way to various applications in epigenetics.

<table border="0" cellpadding="0" cellspacing="0" width="702">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">DMEM&#160;</td>
			<td style="width:145px;">Lonza</td>
			<td style="width:119px;">BE12-614F</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">FBS</td>
			<td style="width:145px;">Invitrogen</td>
			<td style="width:119px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">SILAC DMEM</td>
			<td style="width:145px;">M-Medical</td>
			<td style="width:119px;">FA30E15086</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Dialyzed FBS</td>
			<td style="width:145px;">Invitrogen</td>
			<td style="width:119px;">26400-044</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Lysine 0 (12C6 14N2 L-lysine)</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">L8662</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Arginine 0 (12C6 14N4 L-arginine)</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">A6969</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Lysine 8 (13C6 15N2 L-lysine)</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td>68041</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Arginine 10 (13C6 15N4 L-arginine)</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td>608033</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Micrococcal Nuclease</td>
			<td style="width:145px;">Roche</td>
			<td style="width:119px;">10 107 921 001</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:271px;">Complete, EDTA-free Protease Inhibitor Cocktail Tablets</td>
			<td style="width:145px;">Roche</td>
			<td style="width:119px;">04 693 132 001</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:271px;">Spectra/Por 3 dialysis tubing, 3.5K MWCO, 18mm flat width, 50 foot length</td>
			<td style="width:145px;">Spectrumlabs</td>
			<td style="width:119px;">132720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">QIAquick PCR purification kit</td>
			<td style="width:145px;">QIAGEN</td>
			<td style="width:119px;">28104</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Anti-Histone H3 tri-methylated K9-ChIP grade</td>
			<td>Abcam</td>
			<td>ab8898</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Histone H3 peptide tri-methyl K9&#160;</td>
			<td>Abcam</td>
			<td style="width:119px;">ab1773</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dynabeads Protein G</td>
			<td>Invitrogen</td>
			<td style="width:119px;">100.04D</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">NuPAGE Novex 4-12%&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Bis-Tris Gel&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>NP0335BOX</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Colloidal Blue Staining Kit</td>
			<td>Invitrogen&#160;</td>
			<td>LC6025</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">LDS Sample Buffer</td>
			<td>Invitrogen&#160;</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Formaldheyde</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">F8775</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Aceti anhydride-d6</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">175641-1G</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Formic Acid</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">94318-50ML-F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Iodoacetamide &#8805;99% (HPLC), crystalline</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">I6125</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">DL-Dithiothreitol</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">43815</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Sequencing Grade Modified Trypsin, Frozen 100ug (5 &#215; 20&#956;g)</td>
			<td>Promega</td>
			<td style="width:119px;">V5113</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Nanospray OD 360&#956;m x ID 75&#956;m, tips ID 8&#956;m uncoated Pk 5</td>
			<td style="width:145px;">Microcolumn Srl</td>
			<td style="width:119px;">FS360-75-8-N-5-C15</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">&#160; ReproSil-Pur 120 C18-AQ, 3 &#181;m&#160;&#160; 15 % C</td>
			<td style="width:145px;">Dr. Maisch GmbH</td>
			<td style="width:119px;">r13.aq.</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">C18 extraction disk, 47 mm</td>
			<td style="width:145px;">Agilent Technologies</td>
			<td style="width:119px;">1&#8203;2&#8203;1&#8203;4&#8203;5&#8203;0&#8203;0&#8203;4</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Carbon extraction disk, 47 mm</td>
			<td style="width:145px;">Agilent Technologies</td>
			<td style="width:119px;">12145040</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Cation extraction disk</td>
			<td style="width:145px;">Agilent Technologies</td>
			<td style="width:119px;">66889-U</td>
			<td></td>
		</tr>
	</tbody>
</table>

the chrop approach combines chip and mass spectrometry to dissect locus-specific proteomic landscapes of chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

Chromatin immunoprecipitation is a powerful technique used to profile the localization of a protein or a histone modification along the genome. In a proteomics equivalent, ChIP is followed by MS-based proteomics to identify qualitatively and quantitatively the hPTMs, histone variants and chromatin-binding proteins that are immunoprecipitated together with the modification or protein of interest, used as &#8220;bait&#8221;. In the N-ChroP approach, outlined in Figure 1A, native ChIP, in which chromatin is...

Watch this Scientific Journal Video about The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin at JoVE.com

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

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Biology

18.1K Views.  European Institute of Oncology. By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

Video: The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...