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Analysis of Histone Modifications by Mass Spectrometry


Analysis of Histone Modi?cations by Mass Spectrometry
Ana Villar-Garea,1 Lars Israel,2 and Axel Imhof1
Histone Modi?cations Group, Biomedical Center of the Ludwig Maximilians University of Munich, Germany 2 Protein Analysis Unit, Biomedical Center of the Ludwig Maximilians University of Munich, Germany
1

UNIT 14.10

ABSTRACT Histone N-termini undergo diverse post-translational modi?cations that signi?cantly extend the information potential of the genetic code. Moreover, they appear to mark speci?c chromatin regions, modulating epigenetic control, lineage commitment, and overall function of chromosomes. It is widely accepted that histone modi?cations affect chromatin function, but the exact mechanisms of how modi?cations on histone tails and speci?c combinations of modi?cations are generated, and how they cross-talk with one another, is still enigmatic. Mass spectrometry is ideal for the analysis of histone modi?cations and is becoming the gold standard for histone post-translational modi?cation analysis since it allows the quanti?cation of modi?cations and combinations. This unit describes how high-resolution mass spectrometry can be used to study histone post-translational modi?cations. Curr. Protoc. Protein Sci. 51:14.10.1-14.10.14. C 2008 by John Wiley & Sons, Inc. Keywords: histone modi?cations r lysine acetylation r lysine methylation r mass spectrometry r tandem MS r chemical derivatization of amino acids

INTRODUCTION
Histone modi?cations have been shown to play an important role in determining and maintaining a speci?c gene expression pattern in virtually all eukaryotic cells (Jenuwein and Allis, 2001). Most frequently, histone modi?cations are analyzed using speci?c antibodies directed against a particular modi?cation. However, despite being highly sensitive, these methods depend on the generation of speci?c antisera and do not allow a thorough analysis of combinations of modi?cations on the same polypeptide. Four methods are described in this unit for the isolation of histone molecules (Basic Protocol 1) and the subsequent analysis of histone modi?cations using MALDI-TOF mass spectrometry (Basic Protocols 2 and 3) or tandem mass spectrometry (Basic Protocol 4).

ACID EXTRACTION OF HISTONES
This protocol takes advantage of the basicity of histones to obtain an extract highly enriched in these proteins. Nuclei are usually employed as starting material because removal of the cytoplasmic fraction helps to reduce the number of nonhistone proteins in the preparation. However, the extraction can also be performed starting with whole cells. This is especially useful when there is little sample, to minimize losses due to handling. The amount of starting material should be enough to detect the proteins by Coomassie staining of SDS-PAGE gels. For instance, in the case of mammalian cultured cells, 0.2 × 106 cells (or nuclei) are suf?cient; for Drosophila melanogaster cells, starting with 10 times as many cells is recommended. Many different protocols to prepare nuclei can be chosen from the literature. The authors prefer the shortest procedures, to minimize

BASIC PROTOCOL 1

PostTranslational Modi?cation: Specialized Applications

Current Protocols in Protein Science 14.10.1-14.10.14, February 2008 Published online February 2008 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/0471140864.ps1410s51 Copyright C 2008 John Wiley & Sons, Inc.

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the risk of protein degradation and/or alteration of post-translational modi?cation (PTM) patterns. A simple way to prepare nuclei from mammalian cultured cells is described in Talasz et al., 1996 (also see UNITS 4.2, 4.3 & 24.2). A description of the preparation of nuclei from Drosophila melanogaster embryos appears in Bonaldi et al. (2004a). It should be kept in mind that this procedure will not only extract histones from the cells or nuclei, but also other basic proteins, e.g., high mobility group (HMG) proteins. For the extraction the authors regularly employ 0.25 M hydrochloric acid (Tonino and Rozjin, 1966), but others use 0.4 N sulfuric acid (Gurley et al., 1983; Schechter et al., 2007). To concentrate the proteins and to change the buffer, two different procedures can be used: acetone precipitation or dialysis against acetic acid followed by freeze-drying. Dialysis is often preferred because acetone-precipitated material inconveniently cannot always be completely redissolved, resulting in loss of sample. NOTE: Use Milli-Q-puri?ed water in all steps and for making all solutions.

Materials Eukaryotic cells or nuclei preparation 0.25 M HCl 0.1 M acetic acid containing 1 mM dithiothreitol (DTT; added fresh just before use) 20 mM Tris·Cl pH 6.8 (APPENDIX 2E) containing 1 mM DTT (or other reducing agent, added fresh just before use) Thin-tipped pipet or Dounce homogenizer Shaking incubator or rotator, 4? C Dialysis bags (MWCO 6 to 8 kDa) Extract soluble proteins 1. Centrifuge the cells or nuclei preparation under conditions appropriate for the starting material.
Typical centrifugation conditions are, e.g., 10 min at 500 × g, room temperature, for Drosophila melanogaster cells; 5 min at 380 × g, room temperature (or 5 min at 690 × g, 4? C), for mammalian cultured cells.

2. Decant the supernatant and resuspend the pellet in 0.25 M HCl. Dissociate clumps with repeated pipetting using a thin-tipped pipet or with a few strokes in a Dounce homogenizer until the suspension is homogeneous.
The volume of the acid solution added should be adjusted depending on the amount of cells and the type. For example, for mammalian cells (or the corresponding nuclei), use 0.1 ml acid solution per 106 cells; for nuclei from Drosophila melanogaster embryos, use 1 ml per gram of dechorionated embryos.

3. Rotate or shake the suspension overnight at 4? C. 4. Centrifuge to remove the insoluble material 5 min at 20,800 × g, 4? C. 5. Transfer the supernatant to a clean tube and maintain at 4? C. 6. To ensure a complete extraction, resuspend the pellet in 0.25 M HCl (half of the volume of the acid solution used in step 2) and rotate or shake an additional 3 to 4 hr at 4? C. 7. Centrifuge to remove the insoluble material 5 min at 20,800 × g, 4? C, and combine the supernatant with the one from step 5.
At this point, if necessary, samples can be stored overnight at ?20? C.

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Dialyze extracts 8. Place the pooled supernatants in a dialysis bag and dialyze 1 to 2 hr at 4? C against 0.1 M acetic acid containing 1 mM DTT. Use 0.2 liters dialysis buffer per 1 ml acid extract.
Add DTT immediately before using the buffer.

9. Repeat step 8 twice, using fresh buffer.
The last dialysis can be performed overnight without detectable loss in the quality of the preparation. At this point, samples can be stored up to several days at ?20? C or ?80? C.

Dry and reconstitute samples 10. If there is insoluble material, before proceeding to step 11 centrifuge the extract 5 min at 20,800 × g, 4? C, and discard the pellet.
Depending on the starting material used for the extraction, some precipitate may appear during the dialysis.

11. Freeze-dry the samples. 12. Immediately after freeze-drying, reconstitute the samples in 20 mM Tris·Cl, pH 6.8, containing 1 mM DTT (or similar reducing agent, e.g., TCEP, β-mercaptoethanol).
It is very important that the histones are reconstituted immediately after freeze-drying to prevent the formation of insoluble aggregates. The authors usually employ half of the volume used for the ?rst acid extraction. For instance, with mammalian cells, 50 ?l per 106 starting cells (or nuclei).

13. If there is insoluble material, clear the extract by centrifuging 5 min at 20,800 × g, room temperature. 14. Use immediately or store the reconstituted histones up to several months at ?20? C.

HISTONE ACYLATION AND DIGESTION WITH TRYPSIN
Histones are proteins with a high content of lysine and arginine residues. Conventional trypsin digestion therefore yields very small fragments that are dif?cult to detect by conventional MALDI-TOF. Additionally, in some cases, several tryptic fragments in the same protein have identical masses, or at least the mass difference is too small to be detected with conventional instruments. However, when compared to other proteases, trypsin has several advantages: high speci?city, low self-digestion rates, the ability to perform complete digestions of proteins embedded in gel pieces, and low cost. Trypsin hydrolyzes the peptidic bond between the carboxyl group of lysine or arginine and the following amino acid (except if it is proline), but when the ε-amino group of the lysine residue is modi?ed (N-acetylation, methylation, etc.), trypsin does not recognize the site. The chemical acylation procedure described for histones in this protocol takes advantage of this by modifying all the lysine residues in the protein. As a result, trypsin will only recognize the carboxyl side of the arginine residues as hydrolysis sites. This produces longer peptides that can be easily detected with conventional MALDI-TOF instruments. Various organic anhydrides can be used to acylate the primary and secondary amino groups in the protein. The most frequently employed are propanoic and deuterated (d6) acetic anhydride. Obviously, the use of acetic anhydride with a normal isotopic distribution should be avoided; otherwise the natural and the chemical acetylations cannot be distinguished.

BASIC PROTOCOL 2

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When analyzing the spectra, it is important to remember that: (1) After the acylation of lysine residues, trypsin will digest only after arginine (similar to the endoprotease Arg-C). (2) The only groups that become acylated are primary and secondary amino groups, which are the naturally unmodi?ed or monomethylated ε-amino groups of lysine residues. The naturally acetylated, dimethylated, and trimethylated amino groups do not react in these conditions. The N-terminal amino group of the protein will react in the same way. (3) The masses of the acylated peptides are larger than the unmodi?ed ones, depending on the acylating agent and how many lysine residues become acylated. NOTE: Use Milli-Q-puri?ed water in all steps and for making all solutions.

Materials Histone samples separated by SDS-PAGE (see Critical Parameters and Troubleshooting) 10 mM ammonium bicarbonate Acetonitrile (HPLC grade)/50 mM ammonium bicarbonate Acetonitrile, HPLC grade Propanoic anhydride or acetic anhydride-d6 0.1 M ammonium bicarbonate 1 M ammonium bicarbonate 50 mM ammonium bicarbonate, ice cold 0.2 ?g/?l sequencing grade trypsin, prepared according to the manufacturer’s instructions Scalpel Shaking incubator, 37? C pH-indicator paper Destain gel pieces 1. Slice each puri?ed histone sample band from the gel, chop it in small pieces (cubes of ?1 mm3 ), and place in a 0.2-ml microcentrifuge tube. Add 200 ?l water to each tube and shake 5 min at 37? C.
2. Remove the supernatant with a pipet, taking care to not to remove the gel pieces.
At this point, the proteins are embedded in the gel. Therefore, the same pipet tip can be used to remove all the supernatants without the risk of cross-contamination.

3. Repeat the wash in step 1, using 10 mM ammonium bicarbonate instead of water.
CAUTION: To prevent an explosive opening of the tube by the internal pressure, open and close the tubes several times after adding the ammonium bicarbonate. The ammonium bicarbonate neutralizes the positive charges in the proteins and the leftover acetic acid employed to destain the gel. As a result, CO2 is released and bubbles appear in the solution. The amount of formed gas depends on the amount of acetic acid left in the gel and in the amount and sequence of the protein.

4. Remove the 10 mM ammonium bicarbonate and add 200 ?l of 50% acetonitrile/50 mM ammonium bicarbonate to the gel pieces. Shake 30 to 60 min at 37? C.
This solution will dissolve the dye, and the supernatant will turn blue.
Analysis of Histone Modi?cations by Mass Spectrometry

5. Remove the supernatant. If the gel pieces are still blue, repeat the ammonium bicarbonate wash; if not, proceed to step 6.

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6. Wash with water twice as in steps 1 and 2.
This step serves to remove the ammonium bicarbonate from the gel. Gel pieces can be stored in water at 4? C overnight. The destaining method is modi?ed from Rosenfeld et al. (1992).

Dehydrate gel pieces 7. Add 100 ?l acetonitrile to the gel pieces and shake 5 min at 37? C.
8. Remove the supernatant.
The supernatant from this step has a volume >100 ?l.

9. Repeat steps 7 and 8. Proceed immediately to step 10.
When the gel pieces turn white and small, they are dehydrated. The dehydration must be performed immediately before the subsequent steps. Otherwise, the gel portions will rehydrate from the environmental moisture.

Perform acylation 10. Add 5 ?l anhydride (propanoic or acetic anhydride-d6) and 10 ?l of 0.1 M ammonium bicarbonate. Open and close the tube several times.
CAUTION: The bicarbonate ions neutralize the protons released in the acylation, which causes the formation of CO2 bubbles and pressure buildup.

11. Add 35 ?l of 0.1 M ammonium bicarbonate and incubate 30 to 60 min at 37? C. Test the pH at 5 min after the start of the incubation by placing 1 to 2 ?l supernatant on a piece of pH-indicator paper and, if necessary, add a few microliters of 1 M ammonium bicarbonate to the reaction until the pH is between 7 and 8.
In order to prevent other functional groups from being acylated, make sure that the pH of the ammonium bicarbonate solution does not markedly deviate from 8. The reaction is very fast and a 30-min incubation is usually suf?cient to achieve complete acylation.

12. Remove the supernatant and wash the samples three times with water as described in Steps 1 and 2.
The samples can be stored at 4? C overnight.

Digest histones 13. Dehydrate the gel pieces as described in steps 7 to 9, and place on ice.
14. Prepare, on ice, a master mix containing 10 ?l of 50 mM ammonium bicarbonate and 1 ?l of 0.2 ?g/?l trypsin for each sample. Prepare a 10% excess to compensate for pipetting losses.
In order to determine which signals in the spectra originated from the degradation of trypsin, a blank reaction (trypsin in 50 mM ammonium bicarbonate) can be run in parallel.

15. Immediately add 11 ?l master mix to each tube with gel and incubate on ice until the gel pieces have absorbed all the supernatant (usually 5 min).
The trypsin penetrates the gel at a temperature that prevents self-digestion of the enzyme.

16. Add 40 ?l of 50 mM ammonium bicarbonate to the pieces and incubate overnight at 37? C.
The peptide products of the digestion diffuse out of the gel into the supernatant, therefore, proceeding from the time of the digestion reaction, care must be taken to prevent the cross-contamination of the samples.

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17. Stop the trypsin digestion reaction by placing the tubes on ice.
Because the peptides diffuse out of the gel, it is possible to detect the digestion products by direct mass spectrometry analysis of the supernatant (see Basic Protocol 3). In case the digestion is not complete (miscleavages detected in the spectrum), add 1 ?l of 0.2 ?g/?l trypsin and incubate for an additional 2 to 4 hr at 37? C. In some cases it is worthwhile to perform an acid extraction on the gel pieces before the acquisition of the spectra (see Support Protocol). The acid extraction increases the recovery of the digestion products, which is important when the amount of sample is limited. Additionally, some peptides may adsorb strongly to the gel or to the wall of the tube. The use of acid solutions and acetonitrile helps to recover them. BASIC PROTOCOL 3

MALDI-TOF ANALYSIS OF TRYPSIN-DIGESTED HISTONES
As discussed in UNIT 16.2, the presence of certain buffer components may interfere with the ionization ef?ciency of the sample. Therefore, a desalting step is recommended to obtain good quality spectra in a reproducible manner. There are different commercial chromatographic resins available for this purpose, e.g., hydrophobic phases (C4, C18), cationic exchange (SCX), metal ions (IMAC), or metal oxides (MOAC). In all cases, the experimental procedure consists of binding the peptides in the sample to the chosen stationary phase, washing the resin to remove undesired chemicals, and eluting the peptides with a few microliters of a buffer compatible with the downstream mass spectrometry method. The selection of the resin depends on the aim of the analysis. The hydrophobic resins bind peptides and proteins as a function of their hydrophobicity. The C18 resin is mostly use for peptides and small proteins, whereas C4 yields better results with larger proteins. The metal oxide resins are employed to select phosphorylated peptides from the sample. The metal ions capture not only phosphorylated peptides, but also acidic ones if the carboxyl groups have not been protected. The cationic exchange resins yield an eluate enriched in positively charged peptides. In the case of chemically acylated histones, this feature can be exploited to increase the detection limit of peptides containing dimethylated and trimethylated lysine residues because the acylated amino groups have a much higher pK value than the amines and therefore cannot easily be protonated in aqueous solutions. NOTE: Use Milli-Q-puri?ed water in all steps and for making all solutions.

Materials Acetonitrile, HPLC grade 0.6% (v/v) tri?uoroacetic acid (TFA), spectroscopy grade α-cyano-4-hydroxycinnamic acid (CHCA; Sigma-Aldrich), mass spectrometry grade Trypsin digest (Basic Protocol 2) containing ?0.1 ?g of histone SCX (strong cation exchange resin) or ?C18 (reversed-phase resin) ZipTips (Millipore) Wetting, equilibrating, and washing solutions: prepared according to the ZipTip manufacturer’s instructions or as indicated Washing solution for SCX tips: 0.1% TFA (without methanol) Eluting solution for SCX tips: 5% (v/v) ammonium hydroxide/30% (v/v) methanol (HPLC grade)
Analysis of Histone Modi?cations by Mass Spectrometry

Bath sonicator MALDI target plate Additional reagents and equipment for performing MALDI-TOF spectrometry (UNIT 16.2)

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Prepare matrix solution and sample 1. Prepare the matrix solution: in a 0.5-ml tube, mix 250 ?l acetonitrile and 250 ?l of 0.6% TFA in water. Add a spatula tip’s worth of CHCA. Sonicate 10 to 20 sec in a bath sonicator.
2. Allow to settle and equilibrate 10 to 15 min before use. Store up to one week at room temperature, in the dark.
The solution is saturated with CHCA; therefore, particulates should be visible after sonication and equilibration. If not, add more CHCA and repeat sonication and equilibration. The matrix solution can be stored up to 1 week at room temperature in the dark, but the chemical noise in the low m/z region of the spectra may increase with time. Therefore, if the region below 800 is of interest, preparation of a fresh solution on the same day of use is recommended.

3. To an amount of crude trypsin digest (before or after the formic acid extraction) that contains ?0.1 ?g of the histone(s) of interest, add enough 0.1% TFA bring the volume to 10 ?l.
The exact amount of protein required may vary with the sensitivity of the spectrometer. It is very important that the pH of the samples be below 4 to obtain good and reproducible binding to the resin. If the aim is to desalt a relatively large volume of a sample containing ammonium bicarbonate, a small volume of 10% TFA (up to 0.5% ?nal concentration in the sample) can be added to ensure that the pH will be appropriate and the volume not too big. If the crude samples are very dilute, up to 20 ?l of the quenched sample can be used in the following procedure. If desired, the sample pH can be checked by placing 0.5 to 1 ?l of the solution on a pH-indicator strip.

Prepare resin columns 4a. For ?C18 ZipTips: Prepare the wetting, equilibrating, and washing solutions according to the manufacturer’s instructions (see http://www.millipore.com/catalogue/ module/c5737). Prepare the eluting solution without matrix.
This typically provides better reproducibility than elution with a matrix-containing solution.

4b. For the SCX ZipTips: Prepare the wetting, equilibrating, and eluting solutions according to the manufacturer’s instructions (see http://www.millipore.com/catalogue/ module/c5737). Use 0.1% TFA (without methanol) as washing solution and 5% ammonium hydroxide/30% methanol as eluting solution. 5. Wet and equilibrate a ZipTip by pipetting the corresponding solutions through the tip. Once the resin in the tip is wet, avoid pipetting air.
Avoid pipetting air to prevent the tip from drying because this can affect the binding and elution of the proteins.

6a. If the sample volume is <15 ml: Apply entire sample to the ZipTip and wash with 10 ?l washing solution to remove nonbinding peptides. 6b. If the sample volume is 15 to 20 ?l: Divide it into two portions. Apply one portion to the ZipTip and wash with 10 ?l washing solution to remove nonbinding peptides. Apply the second sample aliquot and wash with another 10 ?l washing solution. 7. Elute the sample in 2 ?l eluting solution. Immediately deposit the eluate onto the MALDI target plate and allow it to dry by evaporation.
If the eluate is not deposited immediately on the target plate, some evaporation of the solvents may occur. As a result, the composition of the solvent mixture can change and some peptides may precipitate in the tube where the eluate is stored.
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The same procedure can be used to prepare the samples for off-line ESI-MS/MS (see Basic Protocol 4) if MS analyses are required to obtain detailed sequence information of the peptide.

8. Once the sample is dry, apply 1 to 2 ?l matrix solution (see step 1) on top. Allow to dry completely.
The volume of matrix solution depends on the size of the spot. Do not take solid particles from the matrix suspension because they may act as seeds for crystal formation, and thereby in?uence the crystallization process. If desired, the matrix solution can be cleared by centrifugation (5 min at top speed, room temperature, in a benchtop centrifuge) after equilibration and before use.

Acquire and analyze spectra 9. Acquire the spectra.
For an introduction to MALDI-TOF spectrometry, see UNIT 16.2. The size of the expected fragments varies depending on the histone and the species. In the case of H4, the biggest peptide has a mass of near 1700 units, whereas in the case of H2A and H2B, the biggest are over 4000 units. The histone sequences can easily be found in the Histone Sequence Database at the NIH (http://research.nhgri.nih.gov/histones), a compilation of the full-length sequences and other features of histones (core and linker) and proteins containing histone-like folds, retrieved from the major public databases (Mari? o-Ram?rez et al., 2006). n ?

10. Analyze the spectra.
An important issue to consider in the interpretation of the spectra is the changes in the peptide masses due to the acylation. The authors employ in silico digests of the proteins performed with the in-house developed program Manuelito (http://sourceforge.net/projects/manuelito). The major advantages of this program are its simplicity, the possibility of considering diverse chemical and natural modi?cations of the sample, and the exhaustive search for possible matches between the spectra and the digested proteins. A disadvantage is that it is necessary to have previously identi?ed the histone with the modi?cations that are being analyzed. Whatever software is used to assign the signals, human supervision is necessary to prevent artifacts. To compare the changes in the modi?cation patterns of several samples that have been processed in parallel (e.g., a knockout cell line and its corresponding wild type), it is possible to integrate the isotopic cluster area of the signals corresponding to a given peptide and its modi?ed forms. The authors usually display the measured area as a percent of all modi?ed isoforms of the peptide of interest. Except in the case of the acetylation (see below), the contribution of a speci?c modi?cation to the total area of the signals for a given peptide is not the true proportion of that modi?cation in the sample. This is due to the fact that two identical peptide sequences carrying different PTMs are chemically different entities. Among other properties, binding to the desalting resin, losses due to nonspeci?c binding to the wall of the tubes, the ionization ef?ciency, and the stability of the ions in the spectrometer may differ between two differentially modi?ed, but otherwise identical, peptides. Therefore, this quantitation method is only useful for sample comparison purposes. In the case of acetylation, if histone acylation has been performed with deuterated acetic anhydride, the modi?ed (naturally acetylated) and unmodi?ed (chemically acetylated) forms of the peptide have almost identical chemical properties, including binding af?nity for the resin employed for desalting or ionization. Because both peptides are in the same sample, both will be affected in the same manner by the same experimental conditions, and one can therefore assume that the ratio of the signals for the naturally acetylated and chemically acetylated (deuterated) forms re?ects the ratio of the acetylated and unmodi?ed species in the sample. In the authors’ hands, the proportion of the signals for acetylated and nonacetylated peptides for a given sample is extremely reproducible between technical replicates (from
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the SDS-PAGE to the MALDI-TOF analysis). The proportions of methylated and nonmethylated forms of a given peptide are more sensitive to the conditions of handling, but, nevertheless, when performing technical replicates to compare two different samples, the trend remains the same.

ANALYSIS OF MODIFIED HISTONE PEPTIDES BY TANDEM MS (MS/MS)
When a single peptide contains multiple modi?able residues, the position cannot be deduced from the determination of the mass alone. Therefore, tandem MS (MS/MS) must be employed to map the sites of modi?cations. This protocol describes the use of a quadrupole-TOF hybrid mass spectrometer for the MS/MS analysis of histone peptides. NOTE: Use Milli-Q-puri?ed water in all steps and for making all solutions.

BASIC PROTOCOL 4

Materials Trypsin digest (Basic Protocol 2) containing ?1 ?g of histone Formic acid, analysis grade ?C18 ZipTips (Millipore) 50% (v/v) methanol/0.1% (v/v) formic acid Ammonium hydroxide GELoader tip (Eppendorf, or similar with extended tips) Needle holder (see Fig. 14.10.1), optional Nano-electrospray capillaries, 1 ?m internal diameter (e.g., Protana) Additional reagents and equipment for desalting peptides (Basic Protocol 3) and analyzing peptides using nanospray techniques and mass spectroscopy (UNIT 16.8)
1. Desalt the trypsin digest as described in Basic Protocol 3, steps 1 to 6, replacing TFA with formic acid in all the solutions. 2. If using C18 ZipTips, elute the sample with at least 2.5 ?l 50% methanol/0.1% formic acid.
Once the sample is eluted, use it immediately to minimize changes in the composition of the solvent mixture due to evaporation. The authors usually employ four to eight times more protein for ESI-MS/MS than for MALDI-TOF.

3. Load the eluate on a nano-electrospray capillary as described in UNIT 16.8.
Touch with the ?ngers only the part of the needle that will be discarded. Use only forceps to handle the tips. The authors employ a homemade needle holder (see Fig. 14.10.1) to place the needle in a benchtop centrifuge and spin it for 3 to 5 sec to remove the air bubbles. If such a device is not available, one can pipet the sample into the capillary and vigorously shake it from up to down, like an oral thermometer.

4. Place the needle in the electrospray source and start the spray (see UNIT 16.8). 5. Optimize voltage, needle position, nitrogen ?ow in the source, and curtain gas ?ow for the maximum intensity.
The values of these parameters depend on the spectrometer and the nature of the sample and solvent.

6. Acquire the spectra.
The ?rst spectrum to be registered should be a simple MS to check which peptides with which charge are present in the mixture. The relative intensities of the signals usually vary

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Figure 14.10.1 Needle holder for nano-electrospray capillary. The sample is eluted from a ZipTip (Millipore) into a GELoader tip (Eppendorf), which is inserted in a nano-electrospray capillary. The assembly is centrifuged 3 to 5 sec in a conventional benchtop centrifuge to load the capillary. Reprinted from an article published in Methods in Enzymology, Vol. 377, Bonaldi, T., Regula, J.T., and Imhof, A., The Use of Mass Spectrometry for the Analysis of Histone Modi?cations, pp. 111-130, copyright 2004, with permission from Elsevier.

with respect to the ones in the MALDI spectra because of differences in the ionization mechanism. The authors usually acquire each spectrum (MS or MS/MS) for 5 min. However, depending on the concentration of the sample and the spectrometer, it may be possible to obtain good quality spectra with shorter acquisition times. The authors manually set the collision energy for the product ion spectra. Usually we vary it during acquisition to obtain as many signals as possible (bigger fragments with low energy, smaller fragments with higher energy). The optimal range of collision energies should be determined empirically as it varies from peptide to peptide. Our recommendation is to acquire the product ion spectra for the unmodi?ed peptides for several reasons. First, it constitutes a control for the presence or absence of certain ions obtained using speci?c experimental conditions. Second, it helps in the interpretation of the spectra of the modi?ed versions of the same peptide. Finally, after the acylation of the samples, peptides carrying natural PTMs may have the same mass as the unmodi?ed peptides and, therefore, it should be determined which forms are present in the sample (only unmodi?ed, only modi?ed, or a mixture).

7. Manually analyze the spectra as described in UNIT 16.11.
SUPPORT PROTOCOL
Analysis of Histone Modi?cations by Mass Spectrometry

FORMIC ACID EXTRACTION OF PEPTIDES AFTER IN-GEL DIGESTION
After the digestion of the proteins in the gel pieces, a certain amount of the crude reaction remains in the gel pieces. If the amount of sample is limiting, it may be worthwhile to recover the residual peptides in the gel. Additionally, some peptides may strongly adsorb onto the polyacrylamide or precipitate in the gel, depending on the buffer employed for

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the digestion. This protocol describes a method for recovering these gel-bound peptides using different solvent mixtures at different pH values. NOTE: Use Milli-Q-puri?ed water in all steps and for making all solutions.

Material Trypsin digest supernatant from gel and gel pieces (Basic Protocol 2) 50 mM ammonium bicarbonate 50% (v/v) acetonitrile/25 mM ammonium bicarbonate 5% (v/v) formic acid 50% (v/v) acetonitrile/2.5% (v/v) formic acid Acetonitrile, HPLC grade 0.1% (v/v) tri?uoroacetic acid (TFA), spectroscopy grade 0.5-ml low protein-binding plastic tube
1. Place the supernatant from each digestion in a clean, 0.5-ml low protein-binding plastic tube. 2. Wash the gel pieces with 50 ?l (or more if necessary to completely cover the gel pieces) of the following solutions in the order indicated, shaking the sample 15 min at 37? C or room temperature:

50 mM ammonium bicarbonate 50% acetonitrile/25 mM ammonium bicarbonate 50 ?l 5% formic acid 50% acetonitrile/ 2.5% formic acid acetonitrile.
Pool the resulting supernatants with the one in step 1.
Some investigators employ TFA instead of formic acid (see UNIT 16.4).

3. Evaporate the pooled supernatants to dryness in a SpeedVac without heating (to prevent undesired reactions). 4. Reconstitute the sample in an adequate volume 0.1% TFA.
To determine how much 0.1% TFA is adequate, the amount of protein and the sensitivity of the subsequent analytical steps must be considered. For instance, when 1 ?g material obtained after applying Basic Protocol 1 to human or Drosophila melanogaster nuclei is loaded on the gel, it should be reconstituted in 10 ?l 0.1% TFA. A 5-?l portion of this solution is suf?cient to obtain good quality MALDI-TOF spectra (see below for details). For a good off-line ESI-MSMS, four to eight times more protein than for the MALDI and reconstituting the peptides in 10 ?l 0.1% formic acid are recommended.

COMMENTARY Background Information
Histones are very basic proteins with isoelectric points above 10. They are found in virtually all eukaryotes and are associated with most of the nuclear DNA. The DNA is wrapped around an octamer formed by the four core histones H2A, H2B, H3, and H4 to build a nucleosome, which is the fundamental repeating unit of chromatin. Since the crystal structures of the octamer and nucleosome have been solved at high resolution (Luger et al., 1997), it is known that the C-terminal globular domains of the core histones form the proteinaceous core of the nucleosome. The N-termini (the tails), in contrast, protrude into solution and do not adopt a de?ned structure that can be resolved by X-ray crystallography. Despite the fact that they do not contribute to the structure of the nucleosome, the sequences of the N-terminal tails are extremely well conserved, suggesting an important role in nucleosome function. This notion is further

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supported by the fact that the histone tails are essential for viability in yeast. The likely reason why these tails have been conserved during evolution is their intense post-translational modi?cation (PTM). The histone H3 tail, for example, is formed by about 38 amino acids, with 19 of them being potentially modi?ed. This not only generates a very high density of PTMs on a short stretch of the protein, but it also produces a challenge for the analysis of modi?cation patterns within this stretch. Histone tails on the nucleosome are subject to enzyme-mediated post-translational modi?cations of selected amino acids, such as lysine acetylation, lysine and arginine methylation, serine phosphorylation, and attachment of the small peptide ubiquitin (Jenuwein and Allis, 2001). These modi?cations, singly or in combination, are thought to generate an epigenetic code that speci?es different patterns of gene expression and silencing (Jenuwein and Allis, 2001). Such information is essential for cells to establish and remember speci?c programs of gene expression, set during embryonic development, corresponding to different cell types.

Critical Parameters and Troubleshooting
The acid extraction of histones from nuclei or whole cells is a very robust protocol. However, sometimes, due to the nature of the starting material, two extractions with hydrochloric acid are not suf?cient to release all the histones from the chromatin, and further washes of the insoluble fraction with acid are necessary. This can be easily monitored by SDSPAGE. The proportion of nonhistone proteins in the extract depends on the starting sample. In Coomassie-stained SDS-PAGE gels, the extracts from nuclei show almost exclusively histones and high mobility group (HMG) proteins, whereas in the extracts from frozen tissues many other bands are visible. In general, however, the predominant signals are due to the core histones. Histones obtained by acid extraction can be separated by HPLC or by acrylamide gel electrophoresis. Each method has its own advantages. Gel electrophoresis affords higher throughput, allowing the puri?cation of several samples in parallel in a single run of about 1.0 to 1.2 hr. Additionally, the sensitivity of the gel staining procedures is usually higher than that of UV detection. However, after electrophoresis the samples are embedded in the gel matrix, which may complicate the accessibility of certain proteases and, therefore, the

Analysis of Histone Modi?cations by Mass Spectrometry

yield of the digestion will be lower. In contrast, the HPLC puri?cation requires a run of about 1 to 1.2 hr per sample, and samples have to be processed sequentially. In this unit, the samples are puri?ed by gel electrophoresis. Detailed protocols for HPLC separation can be found in Lindner et al. (1986) and Gurley et al. (1983). The acylation of proteins in solution is described in Garc?a et al. (2007). ? To separate the histones, samples are electrophoresed in 6-cm (length of the separating gel) SDS-PAGE minigels containing 18% acrylamide, using the L¨ emmli a method (see UNIT 10.1). Nevertheless, histones separated through other sorts of gel electrophoresis—e.g., acetic acid-urea-Triton (AUT) gels (Chang et al., 1999; Kim et al., 2002) or two-dimensional electrophoresis (UNIT 10.4)—can also be employed. The authors stain the gels with Coomassie Brilliant Blue G-250 (see UNIT 10.5, Basic Protocol 1) and destain it with 10% acetic acid (see UNIT 10.5, Alternate Protocol 1). No microwave-assisted procedures are used so as to prevent undesired chemical reactions of the sample. Due to their high basicity, histones migrate more slowly in SDS-PAGE than other proteins of similar molecular weight. As in all mass spectrometric analyses, samples can become contaminated with keratins due to inappropriate handling. To prevent contamination, using a clean (or even new) dish for staining and destaining the gel is strongly recommended. Only touch the gel if it is absolutely necessary and only when wearing new, clean gloves. Use of fresh staining solution every time is recommended. Do not employ any tissue or paper pad to absorb the Coomassie dye during the destaining. After cutting each band, rinse the scalpel thoroughly with water and methanol. Some authors recommend using a slice of the gel where there is no detectable protein to use as a blank control. When carrying out the Support Protocol (formic acid extraction), conduct a careful analysis of the spectra. It has been reported that high concentrations of formic acid can lead to formylation of the peptides (Mak et al., 2001; Pesavento et al., 2007). As a result, their mass increases 27.9949 units, which is indistinguishable from dimethylation (28.0313) in many spectrometers. Evidence for this spurious modi?cation is the appearance in the spectrum of many “dimethylated” peptides, even for peptides where modi?cation has not yet been reported. The authors have not detected

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Supplement 51 Current Protocols in Protein Science

Figure 14.10.2 MALDI-TOF spectrum of histone H4. The sample was prepared from mouse cell nuclei as described in this unit, employing d6-acetic anhydride as an acylating agent. The inset shows a magni?cation of the region where the signals for the peptide 4-17 (GKGGKGLGKGGAKR) appear. The cluster at 1450.89 Da corresponds to the naturally unmodi?ed form; 1447.87 to the naturally monoacetylated species, 1444.8 to the naturally diacetylated peptide, and 1441.84 to the triacetylated peptide.

this phenomenon when working according to the Support Protocol with formic acid extractions, but if doubts arise, use TFA instead of formic acid.

Anticipated Results
As mentioned above, a signi?cant proportion of the histones in each cell carry at least one PTM. Therefore, it is highly likely that PTMs will be detected in any given histone sample (A typical spectrum for an H4 molecule is shown in Fig. 14.10.2.) However, the relative abundances of the different possible modi?cations for a certain histone depend on the nature of the sample (e.g., organism, differentiation status, cell-cycle phase). It should also be kept in mind that the dynamic range of detection for the mass spectrometers is not very high. In other words, peptides of very low abundance may not be detected with the approach described in this unit. For instance, the dynamic range of detection for MALDITOF is about two to three orders of magnitude, which means that peptides with signal of <1% to 0.1% of the most intense signal in the spectrum will not be detected. In that case, other strategies should be employed to analyze the PTMs, e.g., fractionation of the tryptic digest by HPLC enrichment of the desired modi?cation before acquiring the mass spectrum, antibody-based analysis. Trimethylated lysine residues have a very similar mass to acetylated residues (differing
Current Protocols in Protein Science

by only 0.036 units) and because of that, in principle, they can only be distinguished by using extremely high-resolution spectrometers. However, in many cases, when the peptide has a trimethylated lysine residue, a neutral loss of 59 (trimethylamine) will be observed in the MS/MS spectrum of the parent ion. This is not observed in case of acetylation (Zhang et al., 2004). Another difference between a trimethylated and acetylated lysine residue is the production of immonium ions. The immonium ions of lysine tend form ring structures in the spectrometer in such a way that a six-member ring is formed and one of the amino groups (α or ε) is eliminated. The acetylated residues can undergo both types of cyclization, which is re?ected in the spectra by the presence of ions at m/z values of 84 (loss of ε-acetamido group) and 126 (loss of the α-amino group). However, the trimethylated residues can only cyclize if the ε-amino group is lost, which leads to the presence of a single signal at an m/z value of 84 (Zhang et al., 2004).

Time Considerations
The whole process as described in these protocols takes at least 4 days. The hydrochloric acid extraction of the eukaryotic cells or nuclei, which takes place overnight, is started on the ?rst day. On the next day it is possible to complete the acid extraction, dialyze the extracts, and begin the overnight lyophilization.

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The third day is used to separate the proteins in the sample by SDS-PAGE, stain and destain the gel, excise the bands of interest, acylate them, and begin the overnight trypsin digest. Samples are desalted and mass spectra are acquired on the fourth day. It is also possible to acid-extract the peptides from the gel pieces, which adds another half day. Depending on the number of samples, the sort of analysis (study of a speci?c modi?cation or detailed analysis of all the modi?cations present in all the samples), and the spectrometer, the acquisition of ESI-MS/MS spectra may require one additional day. However, as mentioned above, there are points where the process can be stopped for some days and steps that, if necessary, can be performed overnight (e.g., the dialysis), which alters the total time required.

Kim, J.Y., Kim, K.W., Kwon, H.J., Lee, D.W., and Yoo, J.S. 2002. Probing lysine acetylation with a modi?cation-speci?c marker ion using high-performance liquid chromatography/electrospray-mass spectrometry with collision-induced dissociation. Anal. Chem. 74:5443-5449. Lindner, H., Helliger, W., and Puschendorf, B. 1986. Histone separation by high-performance liquid chromatography on C4 reverse-phase columns. Anal. Biochem. 158:424-430. Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., and Richmond, T.J. 1997. Crystal structure of the nucleosome core particle at ? 2.8-A resolution. Nature 389:251-260. Mak, P., Szewczyk, A., Mickowska, B., Kicinska, A., and Dubin, A. 2001. Effect of antimicrobial apomyoglobin 56–131 peptide on liposomes and planar lipid bilayer membrane. Int. J. Antimicrob Agents. 17:137-142. Mari? o-Ram?rez, L., Hsu, B., Baxevanis, A.D., and n ? Landsman, D. 2006. The Histone Database: A comprehensive resource for histones and histone fold-containing proteins. Proteins. 62:838-842. Pesavento, J.J., Garcia, B.J., Streeky, J.A., Kelleher, N.L. and Mizzen, C.A. 2007. Mild performic acid oxidation enhances chromatographic top down mass spectrometric analyses of histones. Mol. Cell. Proteomics In press. Rosenfeld, J., Capdevielle, J., Guillemot, J.C., and Ferrara, P. 1992. In-gel digestion of proteins for internal sequence analysis after one- or twodimensional gel electrophoresis. Anal. Biochem. 203:173-179. Shechter, D., Dormann, H.L., Allis, C.D., and Hake, S.B. 2007. Extraction, puri?cation and analysis of histones. Nat. Protocols 2:1445-1457. Talasz, H., Helliger, W., Puschendorf, B., and Lindner, H. 1996. In vivo phosphorylation of histone H1 variants during the cell cycle. Biochemistry. 35:1761-1767. Tonino, G.J. and Rozijn, T.H. 1966. On the occurrence of histones in yeast. Biochim. Biophys Acta. 124:427-429. Zhang, K., Yau, P.M., Chandrasekhar, B., New, R., Kondrat, R., Imai, B.S., and Bradbury, M.E. 2004. Differentiation between peptides containing acetylated or tri-methylated lysines by mass spectrometry: An application for determining lysine 9 acetylation and methylation of histone H3. Proteomics. 4:1-10.

Literature Cited
Bonaldi, T., Imhof, A., and Regula, J.T. 2004a. A combination of different mass spectrometry techniques for the analysis of dynamic changes of histone modi?cations. Proteomics 4:13821396 Bonaldi, T., Regula, J.T., and Imhof, A. 2004b. The use of mass spectrometry for the analysis of histone modi?cations. In Methods in Enzymology, Vol. 377: Chromatin and Chromatin Remodeling Enzymes (C.D. Allis and C. Wu, eds.) pp. 111-130. Elsevier, London. Chang, L., Ryan, C.A., Schneider, C.A., and Annunziato, A.T. 1999. Preparation/analysis of chromatin replicated in vivo and in isolated nuclei. Methods Enzymol. 304:7699. Garcia, B.A., Mollah, S., Ueberheide, B.M., Busby, S.A., Muratore, T.L., Shabanowitz, J., and Hunt, D.F. 2007. Chemical derivatization of histones for facilitated analysis by mass spectrometry. Nat. Protoc. 2:933-938. Gurley, L.R., Valdez, J.G., Prentice, D.A., and Spall, W.D. 1983. Histone fractionation by high-performance liquid chromatography. Anal. Biochem. 129:132-144. Jenuwein, T. and Allis, C.D. 2001. Translating the histone code. Science 293:1074-1080.

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