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

Summary

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.

Abstract

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.

Introduction

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 wrapped^1,2. All core histones are structured as a globular domain and a flexible N-terminal “tail” 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 histones^3,4. 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 "histone code hypothesis" has gained large consensus in the years but its experimental verification has been held back by technical limitations^6,7.

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 Δmass 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 them^9-14.

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 proteins^16-18 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.

Protocol

1. Cell Culture

1. Standard medium for native ChIP
   1. Grow HeLa cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 1% Glutamine, 1% Pen/Strep and 10 mM HEPES pH 7.5.
2. SILAC labelling for crosslinking ChIP
   1. Grow HeLa cells in SILAC DMEM medium, depleted of lysine and arginine, supplemented with 10% dialyzed FBS, 1% Glutamine, 1% Pen/Strep, 10 mM HEPES pH 7.5 and either the light L-lysine (Lys 0) and L-arginine (Arg 0) or their heavy counterparts, L-lysine (Lys 8) and L-arginine (Arg 10) (Materials Table), at the final concentrations of 73 mg/L and 42 mg/L, respectively.
   2. Grow cells for up to eight generations in SILAC medium to ensure complete isotope-coded amino acids incorporation. Pass cells every two days, when they reach a density of 1.0-1.5 x 10⁶ cells/ml, seeding them at a concentration of 3 x 10⁵ cells/ml.
   3. Evaluate cells growth and viability in SILAC medium to individuate any possible alteration from physiology that may result from the poorer composition of the SILAC medium; to do this:
      1. Visually inspect cells morphology at the microscope every day during the labeling.
      2. Count cells and plot growth curves of cells growing in SILAC versus standard medium.

2. Native Chromatin Immunoprecipitation (N-ChIP)

1. Nuclei preparation from cultured cells
   1. Use 1-2 x 10⁸ unlabeled HeLaS3 cells per experiment. Harvest cells, make aliquot of 50 x 10⁶ cells in 50 ml tubes and centrifuge at 340 x g for 10 min at 4 °C, wash them with ice-cold PBS.
   2. Resuspend each cell pellet in 8 ml Lysis Buffer (Table 1) and incubate for 10 min at 4 °C on a rotating wheel. Pour carefully each cellular lysate on a sucrose cushion (Table 1) and centrifuge in a swing-out rotor, at 3,270 x g for 20 min at 4 °C, to separate nuclei from cytoplasm.
   3. Discard supernatants, keep nuclear pellets and wash them twice in ice-cold PBS by centrifugation, discard supernatant at each wash.
2. Micrococcal Nuclease (MNase) digestion (small scale) and quality control of chromatin after digestion
   1. Resuspend the washed nuclear pellet in 1 ml Digestion Buffer (Table 1); divide in two aliquots of 500 μl and keep on ice.
   2. Measure the Optical Density (OD) at 260 nm of an aliquot, diluted 1:200 in 0.2% sodium dodecyl sulphate (SDS). Note: OD = 1 corresponds to about 50 μg/ml of chromatin DNA. Typically, starting from 2 x 10⁸ cells, the OD is in the range of 40-60.
   3. Take 1% of nuclei, add MNase enzyme to a final concentration of 0.005 U of enzyme/μl of nuclei and incubate at 37 °C for different time lapses (0, 10, 20, 40, 60 min).
   4. At each time point, collect 4 μl of digested nuclei and add 1 mM EDTA to stop the MNase reaction. Keep on ice.
   5. Extract DNA samples by PCR purification kit and elute them in 50 μl TE Buffer (Table 1).
   6. Take 20 μl of each DNA sample, add 10 μl Loading Buffer (Table 1) and load the samples on 1% (w/v) agarose gel containing Ethidium Bromide, with PCR marker as a size control.
   7. Check the digestion evaluating the chromatin nucleosome ladder produced by MNase incubation.
   8. Choose the optimal time for digestion, based on the prevalence of mono-nucleosomes in the preparation. Typically for 0.005 U of enzyme/μl of nuclei the optimal time of MNase digestion is 60 min (Figure 1B, left panel). Note: The optimal MNase concentration/time of digestion may be adjusted depending on the desired cell type and on the size of nucleosomes stretch.
3. Large-scale/preparative MNase digestion and recovery of soluble chromatin fractions
   1. Add to each aliquot (see 2.2.1) 5 μl MNase, corresponding to a final concentration of 0.005 U of enzyme/μl of nuclei; mix gently and incubate at 37 °C for 60 min (or in general for the optimized time interval, based on the small scale test).
   2. Add 1 mM EDTA to stop the reaction and keep on ice. Pellet the digested nuclei by centrifugation at 7,800 x g at 4 °C for 10 min. Transfer the supernatants (fraction S1) in a new tube and store at 4 °C. Note: S1 contains the first soluble fraction of chromatin, comprising mono-nucleosomes. Add the protease inhibitors (Table 1).
   3. Carefully resuspend pellets in 1 ml Dialysis Buffer (Table 1) and dialyze overnight at 4 °C (cut off of the dialysis tube is 3.5 kDa), in 3 L Dialysis Buffer, with constant mild stirring. Collect dialyzed material and centrifuge at 7,800 x g for 10 min at 4 °C. Transfer the supernatant (S2 fraction) in a new Eppendorf tube and store at 4 °C. Note: S2 comprises di- to epta-nucleosomes, depending on the MNase digestion extent.
4. Quality Control of chromatin before immunoprecipitation
   1. Take an aliquot corresponding to 5 μg of S1 and S2 chromatin fractions (quantified by measuring the OD at 260 nm in a NanoDrop system). Extract DNA by PCR purification kit and elute in 50 μl TE Buffer (Table 1).
   2. Take 20 μl of S1 and S2 DNA, mix with 10 μl Loading Buffer (Table 1) and load them on 1% (w/v) agarose gel. Check the quality and efficiency of MNase digestion by visual inspection of the nucleosomes ladder. Note: Fraction S1 is highly enriched in mono-nucleosomes, while fraction S2 contains mainly poly-nucleosomes (Figure 1B, right panel).
5. Incubation of chromatin with antibody
   1. Store at -20 °C 50 μl of S1 fraction for subsequent Mass Spectrometry analysis.
   2. Add 1 volume of ChIP Dilution Buffer (Table 1) to fraction S1.
   3. Add the antibody against the hPTM (or protein) of interest; incubate overnight on a rotating wheel at 4 °C. Note: Typically, use 10 μg to 20 μg of antibody for 2 x 10⁸ cells. The optimal ratio between amount of antibody and starting cells number must be carefully set case by case, depending on the abundance of the hPTM/protein used as bait within the sample and on the antibody efficiency. Optimization is experimental, based on the following tests:
      1. Compare the amount of hPTM/protein of interest between input and flow-through (FT, see 2.7.2) by Western Blot or MS to verify that the immunoprecipitation enriches a significant proportion of specific chromatin region. Note: This is generally achieved when at least 50% of the hPTM/protein bait is depleted in the FT (Figure 1C).
      2. Check that the immunoprecipitated protein of interest is detectable on SDS-PAGE gel, which ensures that a sufficient amount of material is available for the MS analysis. Note: The presence on the gel of the bands corresponding to the four core histones at the correct stoichiometry indicates that the intact nucleosome has been immunoprecipitated by N-ChroP (Figure 1D). Lack of the proper stoichiometry is in fact indicative of a partial disruption/unfolding of the nucleosome.
   4. In parallel, prepare the protein G-coupled magnetic beads (see 2.6).
6. Equilibration and blocking of protein G-coupled magnetic beads
   1. Equilibrate 100 μl of protein G-coupled magnetic beads slurry in Blocking Solution (Table 1), washing three times and incubating them overnight at 4 °C on a rotating wheel. Note: Following the binding capacity of beads, use 100 μl of slurry for 2-20 μg of antibody.
   2. Wash the blocked beads once with Blocking Solution and then twice with ChIP Dilution Buffer (Table 1).
7. Isolation of chromatin using magnetic beads
   1. Add 100 μl of blocked beads to the S1 chromatin sample and incubate them on a rotating wheel at 4 °C for 3 hr. Centrifuge at 340 x g for 1 min to spin down the sample from the lid of the tips. Put in a magnetic rack to pellet the beads.
   2. Transfer the supernatant to a new Eppendorf tube. This is the flow through (FT), namely the fraction of chromatin that did not bind to antibody.
   3. Wash the beads four times in Washing Buffer (Table 1) increasing the salt concentration at each wash (75, 125 and 175 mM NaCl).
   4. To elute the immunoprecipitated chromatin, incubate the beads in 30 μl of LDS Sample Buffer, supplemented with 50 mM dithiothreitol (DTT) for 5 min at 70 °C. Separate the eluted proteins on a 4-12% Bis-Tris acrylamide SDS-PAGE pre-cast gradient gel and stain the gel with Colloidal Coomassie staining kit (Figure 1D).

3. Crosslinking Chromatin Immunoprecipitation (X-ChIP)

1. Crosslinking of cells with formaldehyde
   1. Add 0.75% formaldehyde to SILAC-labeled cells, mix briefly and incubate for 10 min at room temperature. Quench the formaldehyde by adding 125 mM glycine and incubate for 5 min at room temperature.
   2. Divide cells in 5 x 10⁷ aliquots; rinse them three times with ice-cold PBS by centrifuging at 430 x g for 5 min at 4 °C and discarding the supernatants after each wash. At the last wash, discard supernatant and keep the pellet. Note: Once cells are crosslinked, they can be stored at -80 °C, if not immediately used.
2. Nuclei preparation
   1. Resuspend each cell pellet in 10 ml Lysis Buffer (Table 1). Incubate for 10 min at 4 °C with rotation. Centrifuge at 430 x g for 5 min at 4 °C. Discard the supernatants; keep nuclear pellets.
   2. Resuspend each nuclear pellet in 10 ml Washing Buffer (Table 1). Incubate at room temperature for 10 min on a rotating wheel.
   3. Centrifuge at 430 x g for 5 min at 4 °C. Discard the supernatants and collect the pellets.
   4. Resuspend each nuclear pellets in 3 ml ChIP Incubation Buffer (Table 1). Keep on ice.
3. Chromatin sonication and quality control
   1. Sonicate chromatin at 200 W (cycles of 30 sec “on” and 1 min “off”), in a cooled Bioruptor, to fragment chromatin. Note: the number of cycles and sonication intervals depends on the cell type and on the average length of nucleosomal stretch desired. Typically, 30 min of sonication are necessary to generate DNA fragments of 300-500 bp in lengths, corresponding to di- and tri-nucleosomes. The choice of the fragments size depends on the type of chromatin domain under investigation.
   2. Take 2% of total input, reverse the crosslinking by incubation at 65 °C for a minimum of 1 hr in ChIP Incubation Buffer. Extract the DNA by PCR purification kit, elute in 50 μl TE Buffer and load the DNA on agarose gel to check chromatin fragmentation.
4. Incubation of chromatin with antibody
   1. Add 1/10 volume of 10% Triton X-100 to sonicated chromatin. Centrifuge at 12,000 x g for 10 min at 4 °C to pellet debris.
   2. Save 50 μl of chromatin input from heavy cells for testing the incorporation level of SILAC amino acids in labeled cells and for protein profiling.
   3. Add the antibody of choice to the remaining heavy and light labeled sonicated chromatin and incubate overnight at 4 °C on the rotating wheel. In the light channel, add also an excess fold of the soluble peptide. Incubate overnight at 4 °C on a rotating wheel. Notes: The optimal condition between the μg of antibody and the starting material of cells is chosen case by case, as discussed in 2.5.3. The optimal excess fold molarity of the soluble peptide in respect to the antibody must be carefully titrated case by case.
5. Isolation of chromatin using magnetic beads
   1. Equilibrate and block protein G-coupled magnetic beads by following the steps described in 2.6 and using ChIP Incubation Buffer.
   2. Add 100 μl of blocked beads to the chromatin samples and incubate on a rotating wheel for 3 hr at 4 °C. Centrifuge at 340 x g for 1 min to spin down the sample from the lid of the tips. Put in a magnetic rack to pellet the beads. The supernatant is the flow through (FT), containing unbound nucleosomes. Wash beads four times in Washing Buffer (Table 1) with increasing salt concentration (two washes at 150 mM and two at 300 mM NaCl).
   3. Incubate beads in 30 μl SDS-PAGE Loading Sample Buffer (Table 1) and for 25 min at 95 °C to both elute and de-crosslink the immunoprecipitated proteins. Separate proteins on 4-12% Bis-Tris acrylamide SDS-PAGE pre-cast gels (Figure 3D).

4. Sample Preparation Prior to MS

1. In-Gel digestion of histones enriched from N-ChIP
   Note: During protein digestion and peptide extraction steps take care to minimize keratin contaminations that interfere with LC-MS/MS, as previously described^22,23.
   1. Cut gel slices corresponding to the core histones bands (Figure 1D). De-stain the gel pieces with 50% acetonitrile (ACN) in ddH₂O, alternating with 100% ACN to shrink the gel. Repeat until gels pieces are completely de-stained and dry them in a vacuum centrifuge.
   2. Add D₆-acetic anhydride 1:9 (v/v) in 1 M ammonium bicarbonate (NH₄HCO₃) (typically the final volume is 60-100 μl) and 3 μl sodium acetate (CH₃COONa), as catalyzer. Incubate for 3 hr at 37 °C with strong shaking. Note: The sample may generate bubbles in the very first minutes after the assembly of the reaction: it important to handle with caution and release the gas generated, opening from time to time the tubes during incubation.
   3. Rinse the gel pieces several times with NH₄HCO₃, alternate with ACN at increasing percentage (from 50% to 100%), in order to completely eliminate residues of D₆-acetic anhydride.
   4. Shrink the gel pieces in 100% ACN; dry them in a vacuum centrifuge to ensure complete de-hydration. Re-hydrate gel pieces with ice-cold 100 ng/μl trypsin solution in 50mM NH₄HCO₃ and incubate overnight at 37 °C. Note: The combination of chemical modification of lysines using deuterated acetic anhydride and trypsin digestion generates an “Arg-C like” in gel digestion pattern of histones^21,24.
   5. Discard the excess solution and add a volume of 50 mM NH₄HCO₃ to completely cover the gel pieces; incubate overnight at 37 °C.
   6. Collect soluble digested peptides in a new Eppendorf tube. Lyophilize peptides. Resuspend them in 0.5% acetic acid/0.1% trifluoracetic acid (TFA). Desalt and concentrate peptides on a reversed phase C₁₈/Carbon “sandwich” and ion-exchange chromatography (SCX) on hand-made microcolumns (StageTip) ²⁵.
   7. Prepare the StageTip microcolumns by putting Teflon meshwork disks containing immobilized C₁₈/Carbon (“sandwich”) and SCX beads in a 200 μl tips. Obtain the “sandwich” by loading the C₁₈ microcolumn on top of a second tip loaded with Carbon filter. Note: The very short peptides, that are not retained on the C₁₈ filter, pass in the flow-through that is loaded directly on the Carbon Tip, which typically captures them. SCX StageTips can enrich specific peptides, such as H3(3-8) peptide, not efficiently retained by reversed phase chromatography.
   8. Load 50% of peptides onto the C₁₈/Carbon “sandwich StageTip” and 50% onto SCX StageTips. Elute them from the C₁₈/Carbon Tips using 80% ACN/0.5% acetic acid and from the SCX Tips with 5% ammonium hydroxide (NH₄OH)/30% methanol. After lyophilization, resuspend peptides in 0.1% FA and analyze by LC-MS/MS.
2. In-gel Digestion of immunopurified proteins
   In-gel digestion of proteins is carried out as previously described ²², with minor modifications.
   1. Cut each lane in ten slices (Figure 3D) and each slice in small cubes of 1 mm³. De-stain the gel pieces with 50 mM NH₄HCO₃/50% ethanol and add absolute ethanol to shrink the gels. Repeat until the gels are completely de-stained.
   2. Add reduction buffer (Table 1) to the gel pieces for 1 hr at 56 °C, followed by the addition of alkylation buffer (Table 1) for 45 min at room temperature, in the dark. Wash and dry the gel pieces in vacuum centrifuge.
   3. Rehydrate the gel pieces with ice-cold 12.5 ng/μl trypsin solution in 50 mM NH₄HCO₃ and incubate on ice till complete rehydration of the gel pieces. Remove trypsin in excess.
   4. Add 50 mM NH₄HCO₃ to completely cover the gel pieces. Incubate overnight at 37 °C.
   5. Collect the liquid part. Add the extraction buffer (Table 1) to the gel pieces; incubate with strong agitation for 10 min at room temperature. Repeat twice.
   6. Incubate the gel pieces in ACN for 10 min with strong agitation. Repeat twice and pool all supernatants.
   7. Lyophilize the peptides. Resuspend dried peptides in 0.5% acetic acid/0.1% TFA.
   8. Desalt and concentrate peptides on a reversed phase C₁₈ StageTip, as described^26,27.
   9. Elute peptides from the C₁₈ StageTip using 80% ACN/0.5% acetic acid. Remove the organic solvent by evaporating in a vacuum centrifuge and resuspend the peptides in 0.1% FA (typically 5-10 µl), when ready to the MS analysis.

5. LC-MS Analysis

1. Liquid chromatography analysis
   1. Pack the analytical column in a 15 cm fused silica emitter (75 μm inner diameter, 350 μm outer diameter), using reverse-phase (RP) C₁₈, 3 μm resin in methanol, at a constant helium pressure (50 bar) using a bomb-loader device, as described previously²⁸.
   2. Couple the packed emitter (C₁₈ RP column) directly to the outlet of the 6-port valve of the HPLC through a 20-cm long (25 μm inner diameter) fused silica without using pre-column or split device.
   3. Load the digested peptides in C₁₈ RP column at flow of 500 nl/min mobile phase A (0.1% FA/5% ACN in ddH₂O).
   4. After sample loading, separate the peptides using the following gradients:
      1. Apply 0–40% mobile phase B (0.1% FA/99% ACN in ddH₂O) at 250 nl/min over 90 min followed by a gradient of 40-60% in 10 min and 60-80% over 5 min, for elution of peptides deriving from histones (see 2).
      2. Apply 0–36% mobile phase B at 250 nl/min over 120 min followed by a gradient of 36-60% in 10 min and 60-80% over 5 min, for elution of peptides deriving from immunopurified proteins (see 3).
2. Mass Spectrometry analysis using LTQ-FT-ICR-Ultra mass spectrometer
   1. Operate in a data-dependent acquisition (DDA) mode to automatically switch between MS and MSMS acquisition. MS full scan spectra are acquired, typically in the m/z range from 200-1,650, is acquired with resolution R = 100,000 at 400 m/z. The five (Top5) most intense ions are isolated for fragmentation in the linear ion trap using a collision-induced dissociation (CID) at a target value of 5,000.
   2. Use the parameters listed in Table 2 for the “Tuning” acquisition file.
   3. Set the standard acquisition settings as listed in Table 2.

6. Data Analysis

1. Label free quantification of histone modifications co-enriched in chromatin domains
   1. Convert the acquired raw files to mgf files using Raw2msm software (version 1.10)²⁹.
   2. Search the histone modifications using Mascot Deamon (version 2.2.2), setting the parameters described in Table 3.
   3. On the Mascot output peptide list, remove peptides with score lower than 15 or with more than 5 putative PTMs²⁹. Note: For each single peptide ID, select the peptide with the highest Mascot score and filter out all the other redundant peptides with same ID.
   4. Construct the extracted ion chromatograms (XIC) for each precursor corresponding to every modified peptides, based on the m/z value, using the QualBrowser. Calculate the area under the curve (AUC) for each peak (Figure 2A).
   5. Validate each identified peptides containing modifications by visual inspection of the MS/MS spectra using the QualBrowser (Figure 2B).
   6. Calculate the relative abundance for each modified peptide. Calculate the relative enrichment of each modification in the ChIP-ed material. Note: Relative abundance is calculated as a ratio between the AUC of each specific modified peptide over the sum of AUC of all the modified and unmodified forms of the same peptide, while relative enrichment as a ratio of the relative abundance of the specific modification in the ChIP over the Input (Figure 2C).
2. Quantitative proteomic analysis of proteins co-associated within chromatin domains
   1. For proteins identification and quantification use the MaxQuant package (http: //www. maxquant.org/)³⁰. Configure the built-in search engine “Andromeda” using the AndromedaConfig.exe³¹ and set the search parameters listed in Table 3.
3. Incorporation test
   1. Estimate the degree of incorporation of heavy amino acids into proteins in heavy-labeled chromatin input, using MaxQuant software, as follows:
      1. Set the parameters as described in 6.2 but disabling the re-quantify option.
      2. Calculate the incorporation percentage applying the following formula to non-redundant peptide ratios: Incorporation (%) = ratio (H/L) / ratio (H/L) + 1 × 100 (Figure 3B). Note: Accept only if incorporation >95%.

Results

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 “bait”. In the N-ChroP approach, outlined in Figure 1A, native ChIP, in which chromatin is digested wi...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Lonza	BE12-614F
FBS	Invitrogen	10270-106
SILAC DMEM	M-Medical	FA30E15086
Dialyzed FBS	Invitrogen	26400-044
Lysine 0 (12C6 14N2 L-lysine)	Sigma Aldrich	L8662
Arginine 0 (12C6 14N4 L-arginine)	Sigma Aldrich	A6969
Lysine 8 (13C6 15N2 L-lysine)	Sigma Aldrich	68041
Arginine 10 (13C6 15N4 L-arginine)	Sigma Aldrich	608033
Micrococcal Nuclease	Roche	10 107 921 001
Complete, EDTA-free Protease Inhibitor Cocktail Tablets	Roche	04 693 132 001
Spectra/Por 3 dialysis tubing, 3.5K MWCO, 18mm flat width, 50 foot length	Spectrumlabs	132720
QIAquick PCR purification kit	QIAGEN	28104
Anti-Histone H3 tri-methylated K9-ChIP grade	Abcam	ab8898
Histone H3 peptide tri-methyl K9	Abcam	ab1773
Dynabeads Protein G	Invitrogen	100.04D
NuPAGE Novex 4-12%                            Bis-Tris Gel	Invitrogen	NP0335BOX
Colloidal Blue Staining Kit	Invitrogen	LC6025
LDS Sample Buffer	Invitrogen	NP0007
Formaldheyde	Sigma Aldrich	F8775
Aceti anhydride-d6	Sigma Aldrich	175641-1G
[header]
Formic Acid	Sigma Aldrich	94318-50ML-F
Iodoacetamide ≥99% (HPLC), crystalline	Sigma Aldrich	I6125
DL-Dithiothreitol	Sigma Aldrich	43815
Sequencing Grade Modified Trypsin, Frozen 100 μg (5 × 20 μg)	Promega	V5113
Nanospray OD 360μm x ID 75μm, tips ID 8 μm uncoated Pk 5	Microcolumn Srl	FS360-75-8-N-5-C15
ReproSil-Pur 120 C18-AQ, 3 µm   15% C	Dr. Maisch GmbH	r13.aq.
Carbon extraction disk, 47 mm	Agilent Technologies	12145040
Cation extraction disk	Agilent Technologies	66889-U