MM and EM are PhD students within the European School of Molecular Medicine (SEMM). EM is the recipient of a 3-years FIRC-AIRC bursary (Project Code: 22506). Global analyses of R-methyl-proteomes in the TB group are supported by the AIRC IG Grant (Project Code: 21834).

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<li>Yang, F., Shen, Y., Camp, D. G., Smith, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-pH+reversed-phase+chromatography+with+fraction+concatenation+for+2D+proteomic+analysis%5BTitle%5D)+AND+%22Expert+Review+of+Proteomics%22%5BJournal%5D)">High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis.</a> Expert Review of Proteomics. 9 (2), 129-134 (2012).</li>
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<li>Sylvestersen, K. B., Horn, H., Jungmichel, S., Jensen, L. J., Nielsen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteomic+analysis+of+arginine+methylation+sites+in+human+cells+reveals+dynamic+regulation+during+transcriptional+arrest%5BTitle%5D)+AND+%22Molecular+%26+Cellular+Proteomics%22%5BJournal%5D)">Proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest.</a> Molecular & Cellular Proteomics. 13 (8), 2072-2088 (2014).</li>
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</ol>

The authors have nothing to disclose.

The high confidence identification of in vivo protein/peptide methylation by global MS-based proteomics is challenging, due to the risk of high FDR, with several amino acid substitutions and methyl-esterification occurring during sample preparation that are isobaric to methylation and can cause wrong assignments in the absence of orthogonal MS validation strategies. The substoichiometric nature of this PTM further complicates the task of global methyl-proteomics, but can be overcome with the selective enrichment...

Arginine (R)-methylation is a post translational modification (PTM) that decorates around 1% of the mammalian proteome1. Protein Arginine Methyltransferases (PRMTs) are the enzymes catalyzing R-methylation reaction by the deposition of one or two methyl groups to the nitrogen (N) atoms of the guanidino group of the side chain of R in a symmetric or asymmetric manner. In mammals, PRMTs can be grouped into three classes-type I, type II, and type III-depending on their capability to deposit both mono-methylation (MMA) and asymmetric di-methylation (ADMA), MMA and symmetric di-methylation (SDMA) or only MMA, respectively2,3. PRMTs mainly target R residues located within glycine- and arginine-rich regions, known as GAR motifs, but some PRMTs, such as PRMT5 and CARM1, can methylate proline-glycine-methionine-rich (PGM) motifs4. R-methylation has emerged as a protein modulator of several biological processes, such as RNA splicing5, DNA repair6, miRNA biogenesis7, and translation2, fostering the research on this PTM.
Mass Spectrometry (MS) is recognized as the most effective technology to systematically study global R-methylation at protein-, peptide-, and site-resolution. However, this PTM requires some particular precautions for its high-confidence identification by MS. First, R-methylation is substoichiometric, with the unmodified form of the peptides being much more abundant than the modified ones, so that mass spectrometers operating in the Data Dependent Acquisition (DDA) mode will fragment high-intensity unmodified peptides more often than their lower-intensity methylated counterparts8. Moreover, most MS-based workflows for R-methylated site identification suffer from limitations at the bioinformatic analysis level. Indeed, the computational identification of methyl-peptides is prone to high False Discovery Rates (FDR), because this PTM is isobaric to various amino acid substitutions (e.g., glycine into alanine) and chemical modification, such as methyl-esterification of aspartate and glutamate9. Hence, methods based on the isotope labeling of methyl groups, such as Heavy Methyl Stable Isotope Labeling with Amino Acids in Cell culture (hmSILAC), have been implemented as orthogonal strategies for confident MS-identification of in vivo methylations, significantly reducing the rate of false positive annotations10.
Recently, various proteome-wide protocols to study R-methylated proteins have been optimized. The development of antibody-based strategies for the immuno-affinity enrichment of R-methyl-peptides has led to the annotation of several hundreds of R-methylated sites in human cells11,12. Furthermore, many studies3,13 reported that coupling antibody-based enrichment with peptide separation techniques such as HpH-RP chromatography fractionation can boost the overall number of methyl-peptides identified.
This article describes an experimental strategy designed for the systematic and high-confidence identification of R-methylated sites in human cells, based on various biochemical and analytical steps: protein extraction from hmSILAC-labeled cells, parallel double enzymatic digestion with Trypsin and LysargiNase proteases, followed by HpH-RP chromatographic fractionation of digested peptides, coupled with antibody-based immuno-affinity enrichment of MMA-, SDMA-, and ADMA-containing peptides. All affinity-enriched peptides are then analyzed by high-resolution Liquid Chromatography (LC)-MS/MS in DDA mode, and raw MS data are processed by MaxQuant algorithm for identificationof R-methyl-peptides. Finally, the MaxQuant output results are processed with hmSEEKER, an in-house developed bioinformatics tool to search pairs of heavy and light methyl-peptides. Briefly, hmSEEKER reads and filters methyl-peptides identifications from the msms file, then matches each methyl-peptide to its corresponding MS1 peak in the allPeptides file, and, finally, searches the peak of the heavy/light peptide counterpart. For each putative heavy-light pair, the Log2 H/L ratio (LogRatio), Retention Time difference (dRT), and Mass Error (ME) parameters are calculated, and doublets that are located within user-defined cut-offs are labeled as true positives. The workflow of the biochemical protocol is described in Figure 1.

<table><tbody><tr><td>Ammonium Bicarbonate (AMBIC)</td><td>Sigma-Aldrich</td><td>09830</td><td></td></tr><tr><td>Ammonium Persulfate (APS)</td><td>Sigma-Aldrich</td><td>497363</td><td></td></tr><tr><td>C18 Sep-Pak columns vacc 6cc (1g)</td><td>Waters</td><td>WAT036905</td><td></td></tr><tr><td>Colloidal Coomassie staining Instant</td><td>Sigma-Aldrich</td><td>ISB1L-1L</td><td></td></tr><tr><td>cOmplete Mini, EDTA-free</td><td>Roche-Sigma Aldrich</td><td>11836170001</td><td>Protease Inhibitor</td></tr><tr><td>Dialyzed Fetal Bovine Serum (FBS)</td><td>GIBCO ThermoFisher</td><td>26400-044</td><td></td></tr><tr><td>DL-Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>3483-12-3</td><td></td></tr><tr><td>DMEM Medium</td><td>GIBCO ThermoFisher</td><td>requested</td><td>&amp;nbsp;with stabile glutamine and without methionine</td></tr><tr><td>EASY-nano LC 1200 chromatography system</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>EASY-Spray HPLC Columns</td><td>ThermoFisher</td><td>ES907</td><td></td></tr><tr><td>Glycerolo</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>HeLa cells</td><td>ATCC</td><td>ATCC CCL-2</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Iodoacetamide (IAA)</td><td>Sigma-Aldrich</td><td>144-48-9</td><td></td></tr><tr><td>Jupiter C12-RP column</td><td>Phenomenex</td><td>00G-4396-E0</td><td></td></tr><tr><td>L-Methionine</td><td>Sigma-Aldrich</td><td>M5308</td><td>Light (L) Methionine</td></tr><tr><td>L-Methionine-(methyl-13C,d3)</td><td>Sigma-Aldrich</td><td>299154</td><td>Heavy (H) Methionine</td></tr><tr><td>LysargiNase</td><td>Merck Millipore</td><td>EMS0008</td><td></td></tr><tr><td>Microtip Cell Disruptor Sonifier 250</td><td>Branson</td><td></td><td></td></tr><tr><td>N,&lt;em&gt;N,N&amp;prime;,N&amp;prime;&lt;/em&gt;-Tetramethylethylenediamine (TEMED)</td><td>Sigma-Aldrich</td><td>T9281</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>GIBCO ThermoFisher</td><td>15140122</td><td></td></tr><tr><td>PhosSTOP</td><td>Roche-Sigma Aldrich</td><td>4906837001</td><td>Phosphatase Inhibitor</td></tr><tr><td>Pierce C18 Tips</td><td>ThermoFisher</td><td>87782</td><td></td></tr><tr><td>Pierce&amp;nbsp; 0.1% Formic Acid (v/v) in Acetonitrile, LC-MS Grade</td><td>ThermoFisher</td><td>85175</td><td>LC-MS Solvent B</td></tr><tr><td>Pierce&amp;nbsp; 0.1% Formic Acid (v/v) in Water, LC-MS Grade</td><td>ThermoFisher</td><td>85170</td><td>LC-MS Solvent A</td></tr><tr><td>Pierce&amp;nbsp; Acetonitrile (ACN), LC-MS Grade</td><td>ThermoFisher</td><td>51101</td><td></td></tr><tr><td>Pierce&amp;nbsp; Water, LC-MS Grade</td><td>ThermoFisher</td><td>51140</td><td></td></tr><tr><td>Polyacrylamide</td><td>Sigma-Aldrich</td><td>92560</td><td></td></tr><tr><td>Precision Plus Protein&amp;nbsp; All Blue Prestained Protein Standards</td><td>Bio-Rad</td><td>1610373</td><td></td></tr><tr><td>PTMScan antibodies &amp;alpha;-ADMA</td><td>Cell Signaling Technology</td><td>13474</td><td></td></tr><tr><td>PTMScan antibodies &amp;alpha;-MMA</td><td>Cell Signaling Technology</td><td>12235</td><td></td></tr><tr><td>PTMScan antibodies &amp;alpha;-SDMA</td><td>Cell Signaling Technology</td><td>13563</td><td></td></tr><tr><td>Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>Sequencing Grade Modified Trypsin</td><td>Promega</td><td>V5113</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>T6508</td><td></td></tr><tr><td>Ultimate 3000 HPLC</td><td>Dionex</td><td></td><td></td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>U5378</td><td></td></tr><tr><td>Vacuum Concentrator 5301</td><td>Eppendorf</td><td></td><td>Speed vac</td></tr></tbody></table>

a mass spectrometry-based proteomics approach for global and high-confidence protein r-methylation analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

The article describes a workflow for the high-confidence identification of global protein R-methylation, which is based on the combination of the enzymatic digestion of the protein extract with two distinct proteases in parallel, followed by HpH-RP liquid chromatography fractionation of proteolytic peptides and immuno-affinity enrichment of R-methyl-peptides with anti-pan-R-methyl antibodies (Figure 1).
The cells were grown in the presence of Methionine, either na...

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A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

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Research

JoVE Journal

Biochemistry

2.3K Views.  European Institute of Oncology IRCCS (IEO). Dit protocol is zeer belangrijk omdat het de state-of-the-art workflow vertegenwoordigt voor de wereldwijde analyse van eiwit arginine methylatie door massaspectrometrie. Dit is de enige methode om R-gemethyleerd eiwit te identificeren en te profileren met een resolutie in één stijl die niet haalbaar is met andere biochemische technieken. In combinatie met het gebruik van PRMT-remmers, waarvan er verschillende in klinische onderzoeken en geneesmiddelen tegen kanker zijn, maakt dit protocol ...

Video: A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Environment

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

Cancer Research

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Proximity Ligation Assay Allows the Detection, Localization, and Quantification of Protein Arginine Methylation in Fixed Tissue

Arginine methylation is emerging as a key post-translational modification involved in a large range of biological processes. Its study in tissue is often limited by the lack of a specific antibody recognizing the target arginine residue. Proximity ligation assay (PLA) was originally developed to study protein/protein interactions. Here, we describe in detail a PLA protocol dedicated to the detection of arginine methylation that we applied to the glucocorticoid receptor (GR). Having previously shown that PRMT5 dimethylates GRs in cells, we used PLA with a pan symmetrical dimethyl antibody and an anti-GR antibody to measure GR methylation in breast tumors. We demonstrate that PLA offers a unique approach to measure arginine methylation of a target protein, even when the site of methylation has not been identified. This technique could be extended to other post-translational modifications where effective pan antibodies are available. Hence, we detail the PLA technology used to detect arginine methylation in fixed tissue using GR as an example.

Proximity ligation assay is a very useful technique to localize and quantify arginine methylation of a given protein when the modified arginine residue is unknown and/or if no specific antibody is available.

Arginine methylation is emerging as a key post-translational modification involved in a large range of biological processes. Its study in tissue is often ...

Histone Proteomic Analysis and Post-Translational Modification Characterization

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Author Spotlight: Enhanced Histone PTM Isomer Identification Through LC-TIMS-ToF MS/MS and PASEF

An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as ...

Chemistry

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Biology

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.

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 ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Presented here is an optimized high-throughput protocol developed with 16-plex tandem mass tag reagents, enabling quantitative proteome profiling of biological samples. Extensive basic pH fractionation and high-resolution LC-MS/MS mitigate ratio compression and provide deep proteome coverage.

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

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