This work was supported by funding from NIH grants (DP2OD007447, R01GM110174 and R01AI118891).

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The authors declare that they have no competing financial interests.

The protocol described here is optimized considering costs, time, and performance. Other preparations are possible, but they have limitations, especially in the case of coupling with MS analysis. For instance, the high-salt extraction protocol can be used to purify histones26 instead of TCA precipitation (section 3). High-salt protocol is intrinsically milder, as it does not use strong acid. This preserves acid-labile PTMs and increases the yield of extracted histones, as TCA precipitation co-precipitates many other chromatin binding proteins. However, high-salt extraction leads to samples containing too concentrated salt for HPLC-MS/MS. In an alternative preparation, histone digestion can be performed without propionylation (section 6 - 8), for instance by reducing trypsin incubation time and the enzyme/substrate ratio27 or using ArgC as digestion enzyme28-30. However, derivatization with propionic anhydride is recommended, as it leads to the generation of more hydrophobic peptides, which are better retained during liquid chromatography.
For chemical derivatization, a variety of organic acid anhydrides have been evaluated and their merits comprehensively discussed18. Nonetheless, propionic anhydride proved to the best compromise between efficiency, minimized side products and improved peptide hydrophobicity. Potentially, propionic anhydride can be purchased in the isotopically labeled form; this allows for multiplexing analysis due to the possibility of mixing multiple samples and discriminate them at the MS level based on the different masses imparted from the heavy label. However, this analysis leads to an increased complexity of the LC-MS chromatogram and reduces the amount of sample that can be injected for each single condition.
In this regard, some critical aspects of the protocol should be highlighted. The following should be used as checklist to find errors in performing the procedure in case negative results are obtained. First, after nuclei precipitation the pellet should be carefully washed with NIB without NP-40 Alternative (section 2.10) until complete removal of the detergent (noticeable by the lack of bubbles during mixing). Failing to do so would compromise histone extraction with acids. Second, after histone precipitation with TCA (section 3.9) washes of the pellet with acetone is crucial. Presence of concentrated acid would harm the following step if propionylation and digestion (section 6.1) are directly performed. It would be not problematic in case histone fractionation is performed (section 5). Third, it is essential that the propionylation reaction is performed quickly (section 6.3 - 6.7). To do so, avoid using the same propionylation mix (propionic anhydride + acetonitrile) for more than 3 - 4 consecutive samples. Additionally, pH is the most important aspect of trypsin digestion (section 7). If not around 8.0 (7.5 - 8.5) the digestion will be ineffective. This can happen, as the sample will be rich in propionic acid at this step. NH4OH can be added until necessary. Also, for researchers familiar with proteomics workflows it will feel normal to acidify the sample to terminate trypsin digestion. This should not be done, as it will jeopardize the following reaction, i.e., propionylation of peptide N-termini (section 8.1). Finally, in the same issue, it is important to remember for data analysis that unmodified peptides are not actually unmodified; all free lysine residues and N-termini will be occupied by propionylation (56.026 Da). Thus, performing extracting ion chromatography of the mass corresponding uniquely to the peptide sequence would lead to no results.
The limitations of the method are mostly related to the inability of detecting combinatorial PTMs, due to the short peptide sequences, and the biases in achieving the true abundance of a modification, due to the fact that peptides in different modified forms might ionize with different efficiencies. The first issue can be solved by combining this technique with a middle-down or top-down approach (reviewed in 16). This type of analysis, even if technically more challenging, is ideal for studying co-existence frequencies of modifications. Moreover, it allows better discrimination of histone variants, which cannot always be achieved with bottom-up since some peptides have the same sequence in different histone variants. The second issue, related to the ionization efficiency, can be solved using a library of synthetic peptides31. This approach ensures a more accurate estimation of the relative abundance of histone PTMs. However, in most experiments, the desired outcome is the relative changes of given modifications between analyzed conditions. In this case, such correction is not necessary, due to the fact that all samples have the same bias.
In conclusion, this protocol allows for the analysis of histone PTMs that can be completed in 3 days using nLC coupled to tandem MS. Comparisons with techniques other than MS, i.e., using antibody based strategies as discussed in the Introduction, are not suitable, as they cannot achieve even nearly this level of throughput. In addition, antibody based techniques do not allow for the discovery of novel modifications, but they are exclusively based on confirming and quantifying predicted marks. We thus speculate that bottom-up proteomics on histone peptides will gain popularity in proteomics laboratories due to the intuitive advantages in knowing the regulation of histone marks, which are protagonists in tuning gene expression and thus affect the regulation of the proteome. Moreover, the protocol described includes recent improvements in the sample preparation and software for data analysis, which make histone analysis more trivial also for laboratories that never experienced characterization of this type of hypermodified peptides.

Epigenetics is defined as the study of heritable changes in gene expression that arise by mechanisms other than altering the underlying DNA sequence1. Epigenetic regulation is critical during development as the organism undergoes dramatic phenotypic changes even though its DNA content does not change. There are several critical components required for proper epigenetic maintenance, including histone post-translational modifications (PTMs), histone variants, non-coding RNAs, DNA methylation and DNA binding factors, each of which affect gene expression through different mechanisms2. For example, while DNA methylation is a highly stable modification that represses gene translation3, histone variants and histone PTMs are much more dynamic and can influence chromatin in a variety of ways4.
Histone PTMs are mostly localized on the N-terminal tails, as they are the most exposed and flexible region of the protein. However, the nucleosome core is also heavily modified compared to average proteins5. Even though histone marks have been extensively characterized in the last decade, many links between known histone marks and their function are still unclear. This is largely due to the fact that most histone PTMs do not work alone, but rather function in tandem with other PTMs (&#34;cross-talk&#34;) to alter a specific process such as transcription6,7. For instance, the combinatorial mark H3S10K14ac on the gene p21 activates its transcription, which would not occur with only one of the two PTMs8. The protein HP1 compacts chromatin by recognizing H3K9me2/me3 and spreading the modification to nearby nucleosomes. However, HP1 cannot bind H3K9me2/3 when the adjacent S10 is phosphorylated9. Acetylation of H3K4 inhibits binding of the protein spChp1 to H3K9me2/me3 in Schizosaccharomyces pombe10. Furthermore, the histone lysine demethylase PHF8 has the highest nucleosome binding efficiency when three PTMs H3K4me3, K9ac, and K14ac are present11. These examples highlight the importance of achieving a global overview of histone PTM changes rather than focusing on single modifications.
The presence of sequence variants also increases the complexity of histone analysis, as histone isotypes generally have highly similar sequences, but often have different roles in chromatin. For example, H2A.x has a C-terminal sequence which is more easily phosphorylated upon DNA damage compared to canonical H2A12, and it is required for inactivation of sex chromosomes in male mouse meiosis13; similarly, CENP-A substitutes canonical histone H3 in centromeres14. Despite their different functions, these variants share a large portion of their amino acid sequence with the respective canonical histone, making it difficult to identify and quantify them separately.
Antibody-based techniques such as western blotting have been extensively adopted to characterize histones. However, antibody-based approaches are limited for the following reasons: (i) they can only confirm the presence of a modification and cannot identify unknown PTMs; (ii) they are biased due to the presence of co-existing marks, which can influence binding affinity; (iii) they cannot identify combinatorial marks, as only very few antibodies are available for such purpose and (iv) they cross-react between highly similar histone variants or similar PTMs (e.g., di- and trimethylation of lysine residues). Egelhofer et al. described that more than 25% of commercial antibodies fail specificity tests by dot blot or western blot, and among specific antibodies more than 20% fail in chromatin immunoprecipitation experiments15. Mass spectrometry (MS) is currently the most suitable analytical tool to study novel and/or combinatorial PTMs, and it has been extensively implemented for histone proteins (reviewed in 16). This is mostly due to high sensitivity and mass accuracy of MS, and the possibility to perform large-scale analyses.
The bottom-up strategy is the most commonly used MS-based proteomics strategy for histone characterization and their PTMs, wherein the intact protein is enzymatically digested into short peptides (5 - 20 aa). This digestion facilitates both LC separation and MS detection. Masses in the range of 600 - 2,000 Da are commonly more easily ionized and identified with higher mass accuracy and resolution than larger masses. MS/MS fragmentation is also improved, as short peptides are generally well-suited for collision induced dissociation (CID). However, histones present a challenge for bottom-up MS as they are highly enriched in basic amino acid residues, namely lysine and arginine. Therefore, trypsin digestion leads to the generation of peptides that are too small for LC retention and unambiguous localization of the PTMs. To circumvent this issue, our protocol includes lysine and peptide N-terminal chemical derivatization17. The use of propionic anhydride is recommended for efficient chemical derivatization as compared to other reagents 18. Such derivatization blocks the &#603;-amino groups of unmodified and monomethyl lysine residues, allowing trypsin to perform proteolysis only at the C-terminal of arginine residues. Derivatized amines cannot exchange protons with the solution and thus the peptides are generally only doubly or triply charged, facilitating MS and MS/MS detection. Moreover, N-terminal derivatization increases peptide hydrophobicity and thus reversed-phase chromatographic retention. Here, we describe the workflow to purify histones and prepare them for PTM analysis via bottom-up proteomics (Figure 1). This strategy achieves quantification of single histone marks and combinatorial marks for histone PTMs that are relatively close in the amino acid sequence.

<table><tbody><tr><td>Trypsin 0.25% EDTA</td><td>Invitrogen</td><td>25200056</td><td>For harvesting cells</td></tr><tr><td>PBS</td><td>Invitrogen</td><td>14200075</td><td></td></tr><tr><td>Tris</td><td>Roche</td><td>77-86-1</td><td></td></tr><tr><td>Potassium Chloride</td><td>Fisher Scientific</td><td>BP366-500</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>Magnesium Chloride hexahydrate</td><td>Sigma</td><td>M9272</td><td></td></tr><tr><td>Calcium Chloride, anhydrous</td><td>Sigma</td><td>C1016</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>BP220-1</td><td></td></tr><tr><td>DTT</td><td>Invitrogen</td><td>15508-013</td><td></td></tr><tr><td>AEBSF</td><td>EMD Millipore Corp</td><td>101500</td><td></td></tr><tr><td>Microcystin</td><td>Sigma</td><td>M4194</td><td></td></tr><tr><td>Sodium Butyrate</td><td>Sigma</td><td>B5887</td><td></td></tr><tr><td>Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100x)&amp;nbsp;</td><td>Fisher Scientific</td><td>78445</td><td></td></tr><tr><td>NP- 40 Alternative</td><td>CALBIOCHEM</td><td>492016</td><td></td></tr><tr><td>Sulfuric Acid, ACS grade</td><td>Fisher Chemical</td><td>7664-93-9</td><td></td></tr><tr><td>Trichloroacetic acid</td><td>Sigma</td><td>T6399</td><td></td></tr><tr><td>Acetone</td><td>Sigma</td><td>179124</td><td></td></tr><tr><td>HCl</td><td>Fisher Chemical</td><td>A144-500</td><td></td></tr><tr><td>Bradford reagent</td><td>Biorad</td><td>500-0006</td><td></td></tr><tr><td>30% acrylamide/bis 29:1 &amp;#8212; 500 ml</td><td>Biorad</td><td>1610156</td><td></td></tr><tr><td>Coomassie</td><td>Fisher Scientific</td><td>20278</td><td></td></tr><tr><td>C18 Column (5 &amp;#181;m) 2.1&amp;nbsp;mm x 250&amp;nbsp;mm</td><td>Grace</td><td>218TP52</td><td></td></tr><tr><td>C18 Column (5 &amp;#181;m) 4.6&amp;nbsp;mm x 250&amp;nbsp;mm</td><td>Grace</td><td>218TP54</td><td></td></tr><tr><td>HPLC grade acetonitrile</td><td>Fisher Chemical</td><td>A955-4</td><td></td></tr><tr><td>HPLC grade water</td><td>Fisher Scientific</td><td>W6 4</td><td></td></tr><tr><td>TFA</td><td>Fisher Scientific</td><td>A11650</td><td></td></tr><tr><td>Ammonium Bicarbonate</td><td>Sigma</td><td>A6141</td><td></td></tr><tr><td>ammonium hydroxide</td><td>Sigma</td><td>338818</td><td></td></tr><tr><td>propionic anhydride</td><td>Sigma</td><td>240311</td><td></td></tr><tr><td>Sequencing grade modified trypsin</td><td>Promega</td><td>PRV5113</td><td>For digesting histones for MS</td></tr><tr><td>Acetic Acid</td><td>Sigma</td><td>49199</td><td></td></tr><tr><td>C18 extraction disk</td><td>Empore</td><td>2215</td><td></td></tr><tr><td>Formic Acid</td><td>Sigma</td><td>F0507</td><td></td></tr></tbody></table>

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.

As an example, we analyzed histones extracted from human embryonic stem cells (hESCs) with and without retinoic acid (RA) stimulation, starting with 200 &#181;l cell pellets. Presence of RA in cell culture leads to ESC differentiation. From the cell pellet, about 50 - 100 &#181;g of histones were extracted, which is more than sufficient to perform multiple LC-MS injections of histone peptides. After derivatization, digestion, and desalting, the samples were loaded onto a 75 &#181;m x 15 cm C18 column (particle diameter 3 &#181;m, pore size 300 &#197;) in serial mode with a high-performance liquid nano chromatography system with microfluidic chips coupled to a hybrid linear trap quadrupole &#8211; orbitrap mass spectrometer. MS acquisition was performed using DIA. In parallel, samples were also analyzed with a DDA method using a nano-flow UHPLC coupled to a hybrid ion trap-orbitrap mass spectrometer (data not shown). In each cycle, one full MS orbitrap detection was performed with the scan range of 290 to 1,400 m/z, a resolution of 60,000 (at 200 m/z) and AGC of 106. Then, data dependent acquisition mode was applied with a dynamic exclusion of 30 sec. MS/MS scans were followed on parent ions from the most intense ones. Ions with a charge state of one were excluded from MS/MS. An isolation window of 2 m/z was used. Ions were fragmented using collision induced dissociation (CID) with collision energy of 35%. Ion trap detection was used with normal scan range mode and normal scan rate with AGC of 104.
Raw MS data were analyzed adopting software for the extraction of precursor and fragment ion chromatograms, namely Skyline23 and EpiProfile22. EpiProfile has been optimized for histone peptides, as it integrates intelligent peak area extraction due to previous knowledge of peptide retention time. On the other hand, Skyline is optimized for DIA analyses, and thus the DIA figures displayed (Figures 4 and 5A) are screenshots from this software. From the extracted ion chromatogram, the area under the curve is retrieved, and this is used to estimate the abundance of each peptide. The area of the chromatographic peak was calculated for the [M + H]+, [M + 2H]2+, and [M + 3H]3+ ions of the same peptide, even though in most cases the [M + 2H]2+ was the prevalent form. This provides the raw abundance of a given modified form of a peptide. In order to achieve the relative abundance of PTMs, the sum of all different modified forms of a histone peptide was considered as 100%, and the area of the particular peptide was divided by the total area for that histone peptide in all of its modified forms.
Histone peptides are present in a variety of isobaric forms (Figure 5). Isobaric peptides, e.g., K18ac and K23ac, can only be quantified at the MS/MS level, where their unique fragment ions are used to determine the ratio of the isobaric species (Figure 5A and 5B). This ratio is used to divide the area of the chromatographic peak between the two species. When using DDA, these isobaric forms were included in a list of targeted masses, because these peptides need to be selected for fragmentation through their entire elution, which would not occur in a standard DDA experiment. The discrimination of the relative abundance of the isobaric species is then performed by monitoring the elution profile of the fragment ions. On the other hand, DIA type of acquisition does not require any inclusion list. However, this type of acquisition method is not compatible with traditional database searching, and thus might prevent the discovery of unknown modified peptides.
Lysine acetylation (+ 42.011 Da) was discriminated from the nearly isobaric trimethylation (+ 42.047 Da) by using high resolution MS acquisition (&#62; 30,000). Moreover, acetylation is more hydrophobic than trimethylation, leading to elution of acetylated peptides later than the respective trimethylated ones. The unmodified form of the same peptide elutes even later, due to the fact that the lysine is propionylated. In summary, the order of hydrophobicity for a peptide with one modifiable site is di- and trimethylated &#60; acetylated &#60; unmodified (propionylated) &#60; monomethylated (propionylated).
hESCs showed a clear reduction of acetylated peptides when stimulated for differentiation (Figure 6A and 6B). This was not surprising, as previous results reported higher acetylation in ESCs as compared to differentiating ones25, reflecting the generally permissive nature of the pluripotent chromatin. By focusing on histone H3, 35 different modified forms were quantified (Figure 6C). However, all histone proteoforms that can be investigated with this approach are more than 200, including all histone variants and low abundance modifications (data not shown). Moreover, our analysis showed that high reproducibility can be obtained between technical replicates, as evidenced by the small size of the error bars (representing &#177; standard deviation). Taken together, this section describes how to extract the relative abundance of histone modified peptides using nLC-MS data.
<img alt="Figure 1" src="/files/ftp_upload/54112/54112fig1.jpg" /> 
Figure 1: Workflow for Bottom-up MS/MS Histone Analysis. The ten steps for histone analysis are shown, including an estimation of the time required for each step. The section number is given in parenthesis as present in the manuscript. Section 5, describing sample fractionation to isolate the various histone variants, can be omitted unless there is a need for highly sensitive analysis of a given variant. <a href="https://www.jove.com/files/ftp_upload/54112/54112fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54112/54112fig2.jpg" /> 
Figure 2: Reversed-Phase High Flow LC for Histone Variant Fractionation and Coomassie Gel. (A) LC-UV chromatogram representing intact histone separation. Histone H3 variants can be discriminated from one another according to their elution time. Fractions can be collected either manually or using an automated fraction collector. (B) Coomassie gel of three replicates of histone purification. <a href="https://www.jove.com/files/ftp_upload/54112/54112fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54112/54112fig3.jpg" /> 
Figure 3: Making of Stage-tipping Plug. With a P1000 pipette tip, punch a disk made of C18 material from a solid phase extraction disk (second panel). The minidisk will stick in the tip (middle panel), so that it can be pushed out into a smaller P100/200 pipette tip using any kind of small capillary. In this example, we used a 700 &#181;m external diameter fused silica tubing. The minidisk should be pushed to the bottom of the P100/200 pipette tip until it cannot go any further (last panel). The stage tip is ready for histone desalting, as it has sufficient capacity to retain enough sample material for numerous replicates. Specifically, one minidisk is enough for 15 - 20 &#181;g of sample. If more sample is required, multiple disks can be packed on one another. <a href="https://www.jove.com/files/ftp_upload/54112/54112fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54112/54112fig4.jpg" /> 
Figure 4: Schematic Representation of DDA and DIA Methods. When using DDA, the MS scan cycle is characterized by sequential selection of precursor ions for MS/MS fragmentation according to their intensity and charge state. Once a precursor ion has been fragmented it is placed into an exclusion list to avoid repetitive selection of the same peptide, so that the MS can &#34;dig&#34; into less abundant signals. This acquisition method is the technique of choice in proteomics for discovery mode. Quantification is achieved by integrating the full scan signal of a given ion next to the identified MS/MS spectrum. In DIA, the entire m/z range is fragmented at every scan cycle. This approach is less suitable for discovery mode, but it produces a chromatographic profile of all ions, precursors and products. This leads to more confident quantification and discrimination of isobaric forms. <a href="https://www.jove.com/files/ftp_upload/54112/54112fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54112/54112fig5.jpg" /> 
Figure 5: Quantification of Isobaric Peptides. (A) Example of two isobaric peptides commonly abundant in histone analysis. The extracted ion chromatogram (XIC) of their precursor mass and relative isotopes (above) is identical. However, the XIC of the product ions (below) allows for discrimination of the two isobaric forms. Notably, only unique fragment ions should be used to estimate the relative abundance of the two species. (B) Representation of the unique fragment ions for the two described peptides (highlighted in red). (C) List of the commonly analyzed peptides in Homo sapiens having at least one isobaric equivalent. Sequence variants between the listed histone peptides are indicated. <a href="https://www.jove.com/files/ftp_upload/54112/54112fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54112/54112fig6.jpg" /> 
Figure 6: Representative Results of Human Embryonic Stem Cells with and without Retinoic Acid Treatment. (A) Relative quantification of the histone H3 peptide KQLATKAAR (aa 18 - 26) in all of its modified proteoforms. The relative abundance was estimated using all proteoforms as 100% (the relative percentage of the unmodified peptide is not shown). (B) Relative quantification of the histone H3 peptide KSTGGKAPR (aa 9 - 17). (C) Relative abundance of detected peptides for canonical histone H3 with and without cell treatment with retinoic acid. The figure indicates in which of the two treatments the given modifications are more abundant (&#62; 50%). Overall, we demonstrate that histone H3 acetylation decreases in most of the lysine residues upon induction of cell differentiation. <a href="https://www.jove.com/files/ftp_upload/54112/54112fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Solution #</td>
			<td colspan="7">Composition</td>
		</tr>
		<tr>
			<td>1</td>
			<td colspan="7">Nuclear Isolation Buffer (NIB) stock is made as follows and stored frozen as 100 ml aliquots at -20 &#176;C; thawed NIB can be stored at 4 &#176;C&#160; for few weeks: 15 mM Tris, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, and 250 mM sucrose. The pH of the buffer is adjusted to 7.5 with HCl.&#160;</td>
		</tr>
		<tr>
			<td>2</td>
			<td colspan="7">Protease inhibitors (add fresh to buffers prior to use): 1 M Dithiothreitol (DTT) in ddH2O (1,000x); 200 mM AEBSF in ddH2O (400x)</td>
		</tr>
		<tr>
			<td>3</td>
			<td colspan="7">phosphatase inhibitor (add fresh to buffers prior to use): 2.5 &#181;M Microcystin in 100% ethanol (500x)</td>
		</tr>
		<tr>
			<td>4</td>
			<td colspan="7">HDAC inhibitor (add fresh to buffers prior to use): 5 M Sodium butyrate, made by titration of 5 M butyric acid using NaOH to pH 7.0 (500x)</td>
		</tr>
		<tr>
			<td>5</td>
			<td colspan="7">NP-40 Alternative: 10% v/v in ddH2O</td>
		</tr>
		<tr>
			<td>6</td>
			<td colspan="7">0.2 M H2SO4 in ddH2O</td>
		</tr>
		<tr>
			<td>7</td>
			<td colspan="7">Trichloroacetic acid (TCA): 100% w/v in ddH2O</td>
		</tr>
		<tr>
			<td>8</td>
			<td colspan="7">Acetone+0.1% Hydrochloric acid (HCl): 0.1% v/v HCl in acetone</td>
		</tr>
	</tbody>
</table>
Table 1. Solutions.

Watch this Scientific Journal Video about Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis at JoVE

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

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 ...

complete-workflow-for-analysis-histone-post-translational

Research

JoVE Journal

Biology

29.0K Views.  University of Pennsylvania.  Dit protocol beschrijft een volledig geïntegreerde workflow voor het karakteriseren histon post-translationele modificaties met massaspectrometrie (MS). De workflow bevat histon zuivering uit celkweken of weefsels, histon derivatisering en de spijsvertering, MS analyse met behulp van nano-flow vloeistofchromatografie en instructies voor gegevensanalyse. Het protocol is ontworpen voor de voltooiing binnen 2-3 dagen. 

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

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

Biochemistry

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Medicine

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. ...

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

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

2D Gel Separation for Histone Isoform Enrichment and PTM Characterization

Unveiling Histone Proteoforms using 2D-TAU Gel Electrophoresis

Histones undergo various post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, acylation, and ubiquitination, which control nucleosome dynamics and determine cell fate. The nucleosome, which is the functional unit of chromatin, comprises DNA, four pairs of histones (H3, H4, H2A, and H2B) making up the globular core, and the linker histone H1, which stabilizes the chromatin structure. The amino (N)-terminal tails of the histones protrude from the globular core domains and undergo distinct PTMs that influence the chromatin landscape. Some evidence suggests that histone PTM homeostasis is crucial for preserving all physiological activities. The deregulation of histone PTMs is the primary cause of abnormal cellular proliferation, invasion, and metastasis. Therefore, developing methods for characterizing histone PTMs is crucial. Here, we describe an effective technique for isolating and analyzing histone isoforms. The method, based on the combination of two orthogonal separations, allows the enrichment of histone isoforms and the following mass spectrometry identification. The technique, originally described by Shechter et al., combines acid-urea polyacrylamide gels (TAU-GEL), which can separate basic histone proteins based on size and charge, and Sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), which can separate proteins by molecular weight. The result is a two-dimensional map of histone isoforms, suitable for in-gel digestion followed by mass spectrometry identification and western blot analysis. The result is a two-dimensional map of histone isoforms, suitable for both in-gel digestion followed by mass spectrometry identification and western blot analysis. This proteomic approach is a robust method that allows the enrichment of a single histone isoform and the characterization of new histone PTMs.

Author Spotlight: Epigenetic Modifications and Metabolic Rewiring as Targets for Cancer Therapy

This method describes a simple and fast strategy for histone proteoform characterization and investigation.

Histones undergo various post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, acylation, and ubiquitination, which ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

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 ...

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.

This protocol outlines how a three-dimensional cell culture system can be used to model, treat, and analyze chromatin modifications in a near-physiological state.

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid ...

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

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