Ultra-High Performance Liquid Chromatography−Mass Spectrometry

Ultra-High Performance Liquid Chromatography-Mass Spectrometry for the Fast Profiling of Histone Post-Translational Modifications Ke´vin Contrepois,† Eric Ezan,‡ Carl Mann,† and Franc¸ois Fenaille*,‡ CEA, iBiTecS, Service de Biologie Inte´grative et Ge´ne´tique Mole´culaire (SBIGeM) and Service de Pharmacologie et d’Immunoanalyse (SPI), F-91191 Gif Sur Yvette, France Received May 19, 2010

Abstract: Histones are subjected to extensive posttranslational modifications (PTMs) that are known to play key roles in many biological processes. In this study, we report a fast, efficient, highly reproducible, and easily automated method involving ultra-high performance liquid chromatography (UHPLC) coupled to a high resolution/high mass accuracy LTQ-Orbitrap mass spectrometer to profile core histone modifications/variants from WI-38 primary human fibroblasts. The whole analysis was performed on intact unfractionated histones within 19 min, which is ∼3-fold faster than previously published procedures. High mass accuracy measurements combined with top-down tandem mass spectrometry (MS) experiments enable accurate histone identification. Experimental and biological variations were thoroughly assessed and were 8% and 16% on average, respectively. With a sample preparation reduced to the minimum, characterization of the most abundant histones can be achieved in a single experiment. Semi-quantitative information can be obtained with respect to the relative abundances of the detected isoforms through a label-free approach. Isoform identities and relative distributions were further confirmed by the LC-MS/MS analysis of tryptic digests. Overall, our UHPLC-MS approach for histone profiling offers a sensitive and reproducible tool that will be of great value for exploring PTMs and variants and can readily be applied to clinical or pharmaceutical studies.

octamer, which comprises pairs of the core histones H2A, H2B, H3 and H4 and/or their variants.1 The DNA is further compacted into a larger chromatin fiber thanks to non-histone proteins and linker histone H1.2 Post-translational modifications (PTMs) on histones play a key role in cellular processes requiring access to the genome, such as DNA transcription,3 replication,4 repair,5 and recombination by influencing transitions between chromatin physical states: open (euchromatin) and closed (heterochromatin) conformations. Histone covalent modifications act either directly or via interacting proteins in this phenomenon.6 Moreover, nucleosomes can transmit epigenetic information from one cell generation to the next.7 Histones are preferentially modified on their flexible N-terminal tails that protrude from the nucleosome. Canonical histones and histone variants are subjected to extensive PTMs including mainly acetylation, mono-, di- and trimethylation of lysine residues.6 Numerous but specific residues can be covalently, heterogeneously and dynamically modified, thus leading to highly complex combinatorial modification profiles. This particular behavior has led to the “histone code” hypothesis,8 which postulates that multiple coexisting histone PTMs (on the same N-terminal tail or not) form combinatorial codes that function to dynamically regulate cellular events via “binding platforms” for specific proteins that “read” the code and translate it into biological responses.9,10

Chromatin, a macromolecular complex composed of DNA and proteins, is the heritable material in eukaryotes. The repeating unit structure of chromatin is the nucleosome; it contains ∼147 base pairs of DNA wrapped around a histone

Although this histone code is not yet completely deciphered, it has been clearly established that these modifications and the enzymes responsible for them play critical roles in carcinogenesis.11 For instance, histone deacetylases (HDACs) are now regarded as being promising targets in drug development for cancer therapy.12 Besides, some particular disease states may influence histone variant relative abundances. For instance, it has been recently shown that the expression of some H2A variants (H2AFL and H2AFA/M*) was changed in chronic lymphocytic leukemia.13 In this context, the development of fast, reproducible and validated analytical approaches for identifying and measuring histone variants and PTM abundances is crucial in understanding their biological functions and assessing the effects of novel pharmacological agents.

* To whom correspondence should be addressed. Phone: 33-1-69-08-7954. Fax: 33-1-69-08-59-07. E-mail: [email protected]. Mailing address: CEA, iBiTecS, Service de Pharmacologie et d’Immunoanalyse (SPI), Building 136, CEA Saclay, F-91191 Gif Sur Yvette, France. † CEA, iBiTecS, Service de Biologie Inte´grative et Ge´ne´tique Mole´culaire (SBIGeM). ‡ CEA, iBiTecS, Service de Pharmacologie et d’Immunoanalyse (SPI).

Mass spectrometry (MS) has rapidly become appreciated by chromatin biologists as a complementary tool to antibodybased approaches for its speed and its ability to assign, quantify or discover new histone modification sites and combinations. Historically, researchers have first developed bottom-up MS approaches, where enzymes such as trypsin were used to digest

Keywords: histones • post-translational modifications • ultra-high performance liquid chromatography • mass spectrometry • label-free quantification

Introduction

10.1021/pr100497a

 2010 American Chemical Society

Journal of Proteome Research 2010, 9, 5501–5509 5501 Published on Web 08/14/2010

technical notes proteins into small peptides prior to analysis by reverse-phase high performance liquid chromatography (RP-HPLC) coupled to different varieties of tandem mass spectrometry (MS/MS). While ideally suited for most proteins, trypsin digestion presents some limitations for studying histones, due to their high content of lysine and arginine residues. Indeed, trypsin digestion of histone proteins often leads to small and irreproducible peptides that are not well retained on classical RP-HPLC columns and are also difficult to analyze by MS.14 As an alternative, lysine amino groups can be first chemically modified by reaction with propionic anhydride, to further generate propionylated residues that would be resistant to trypsin proteolysis. Under these conditions, reproducible and MSfriendly Arg-C-type peptides can be obtained.14 As most of the protocols already described for histone analysis by MS, this chemical derivatization was initially performed on HPLCpurified histones. To further bypass such time- and materialconsuming off-line fractionation steps, Garcia and co-workers recently developed an optimized derivatization procedure compatible with unfractionated total acid-extracted histones.15 While highly efficient in obtaining site-specific information, such bottom-up approaches can be of limited interest to further elucidate global combinatorial codes of histone PTMs that are not in close proximity to each other.16 To encompass this limitation, top-down MS approaches have been developed.17-19 Top-down strategies commonly comprise (i) accurate mass measurement of intact proteins by high resolution and high mass accuracy MS, thus giving access to the connectivity between PTMs, and (ii) subsequent MS/MS acquisition performed with either collisional- or electron-based ion fragmentation on a given protein isoform, to further obtain deeper insights into its primary sequence and PTMs. Direct coupling of RP-HPLC to electrospray ionization (ESI) mass spectrometry (MS-only acquisition) has recently shown great promises for histone characterization.19-21 It basically allows the simultaneous assessment of histone modifications and variants, and their relative abundances can also be determined from a single LC-MS run. Such an approach becomes especially powerful when involving high resolution and high accuracy mass spectrometers such as LTQ-Orbitrap13 or Fourier transform ion cyclotron resonance (FT-ICR)19 instruments. However, the average runtime of these LC-MS methods is >60 min. Thus, faster LC-MS methods, compatible with the high-throughput clinical or pharmaceutical profiling of histone modifications, are still lacking. In the present study, we have developed a fast, efficient and highly reproducible method involving ultra-high performance liquid chromatography (UHPLC) coupled to a high resolution/ high mass accuracy LTQ-Orbitrap instrument for the analysis of core histone modifications/variants. The chromatographic separation was performed using formic acid as mobile phase additive and enables the profiling of core histones posttranslational modifications in a single run of less than 20 min. The reproducibility of the approach has also been assessed by performing independent analytical and biological replicate experiments. The identity and relative distributions of histone isoforms were further confirmed by the LC-MS/MS analysis of tryptic digests.

Materials and Methods Cell Culture, Nuclei Isolation and Histone Extraction. All products used for cell culture were from Gibco (Invitrogen, France). Human WI-38 primary embryonic fibroblasts im5502

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Contrepois et al. mortalized with human telomerase were grown at 37 °C under 5% CO2 and 5% O2 atmosphere in Modified Eagle’s Medium (MEM) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 1 mM non essential amino acids, and 2 mM L-glutamine. Histones were prepared from 20-60 million cells. Cells were individualized for approximately 5 min at 37 °C with an adequate volume of prewarmed 0.25% Trypsin-EDTA reagent (Gibco) to cover the culture surface. After cell detachment, two volume equivalents of ice-cold growth medium were added to neutralize the trypsin before pelleting. As previously described,22,23 cell pellets were subsequently washed with 10 mL of ice-cold phosphate buffer saline (PBS) and 10 mL of icecold hypotonic lysis buffer containing: 10 mM Tris-HCl, 10 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, and adjusted at pH 7.5. This buffer was supplemented with deacetylase inhibitor (10 mM sodium butyrate, Sigma-Aldrich, Saint Quentin Fallavier, France), phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich) and protease inhibitor cocktail (Roche, Indianapolis, IN). Cells were resuspended in approximately 2 mL of ice-cold hypotonic lysis buffer, incubated 10 min on ice and frozen at -20 °C. After thawing, cells were gently lyzed by performing 25 strokes with a Dounce homogenizer. Histones were then extracted from nuclei with 0.4 N H2SO4 during 4 h on a rotator at 4 °C. Histones were precipitated with trichloroacetic acid (30% TCA), followed by three washes with pure acetone. The resulting pellet was reconstituted in deionized water and frozen at -80 °C prior to further processing. Total protein concentrations were determined by Bradford assay and were in the 1.0-1.5 µg/µL range. An aliquot of this protein fraction was checked by SDS-PAGE using Coomassie blue staining. Typical yields were approximately 70 µg total histone from 10 million cells. Mild Performic Acid Oxidation of Unfractionated AcidExtracted Histones. Histones were oxidized by incubating at room temperature 15 µL of protein solution (1.0 µg/µL) with 5 µL of a mixture of 12% (v/v) formic acid and 12% H2O2 (v/v) for 4 h at room temperature (final concentration: 3% formic acid + 3% H2O2), as previously described.24 Unfractionated Intact Histones Analysis by UHPLC-MS. About 10 µg of unfractionated acid-extracted histones (native or oxidized) were loaded on a C18 Hypersil GOLD column (2.1 mm ×150 mm, 175 Å, 1.9 µm, Thermo, San Jose, CA). Native histones were separated at a constant flow rate of 300 µL/min using a linear gradient of 0 to 50% B in 12 min (with solvent A: H2O containing 0.1% formic acid and solvent B: ACN containing 0.1% formic acid). The oven temperature was set at 50 °C. The source parameters were as follows: capillary temperature, 275 °C; source voltage, 4.50 kV; capillary voltage, 49 V; tube lens voltage, 195 V; sheath gas flow, 30 arbitrary units; auxiliary gas flow, 3 arbitrary units. The UHPLC Accela system was coupled to an LTQ-Orbitrap Discovery mass spectrometer (Thermo, San Jose, CA) operating in the full scan mode (m/z 300-1,700) with a resolution of 30,000 (at m/z 400). All data were collected in profile mode. Oxidized histones were analyzed exactly using the same conditions, except that the 0-50% B linear gradient was performed in 13.5 min instead of 12 min. LTQ Orbitrap data were deconvoluted by using the Xtract software included in the Xcalibur package. Relative quantification of histone modified forms/variants was performed by dividing the intensity of a given deconvoluted MS peak by the sum of the intensities of the different deconvoluted MS peaks composing the spectrum of a considered histone.25 Top-down experiments were performed by fragmenting ions in the ion trap using a normalized collision energy of 70%, with an action q of 0.25

technical notes

Fast Profiling of Histone Post-Translational Modifications and an activation time of 30 ms. An isolation width of m/z 70 around the two parent ions at m/z 700 and m/z 1150 was used. Fragment ions were detected in the Orbitrap analyzer at a target resolution of 30,000 at m/z 400. Trypsin Digestion of Histones. Before trypsin digestion, unfractionated acid-extracted histones (∼30 µg in 30 µL of 50 mM ammonium bicarbonate) were chemically derivatized by treatment with propionic anhydride essentially as described before.14 Briefly, this reaction mixture was created using 1/4 propionyl anhydride (Sigma-Aldrich) and 3/4 methanol. Equal volumes of propionylation reagent and histones in 50 mM ammonium bicarbonate were mixed and allowed to react at 37 °C for 15 min and reduced near to dryness using a SpeedVac concentrator for removal of reaction remnants. Propionylated histones were then diluted with 30 µL of ammonium bicarbonate (100 mM, pH 7.8) and further digested with trypsin (Promega, Madison, WI) at an enzyme/substrate ratio of 1:20 for 5 h at 37 °C (i.e., by addition of 1.5 µL from a 1 µg/µL stock solution). The reaction was then quenched by addition of 1 µL of concentrated acetic acid. Samples were then reduced near dryness using a SpeedVac concentrator and resuspended in 15 µL of 50 mM ammonium bicarbonate. A second round of propionylation was then performed to propionylate the newly created peptide N-termini. LC-MS/MS Conditions for the Analysis of Tryptic Digests. Chromatographic separation of enzymatically digested samples was performed on a Zorbax 300SB C18 column (2.1 mm × 150 mm, 300 Å, 5 µm, Interchim, Montluc¸on, France). Mobile phases consisted of 0.1% formic acid in water (A) and ACN containing 0.1% formic acid (B). Peptides were eluted from the column at 300 µL/min using a 0-60% phase B gradient over 120 min. The oven temperature was set at 20 °C. The HPLC system was coupled to an LTQ-Orbitrap Discovery mass spectrometer, operating in the data-dependent acquisition mode as previously described.26 Briefly, the source conditions were as follows: capillary temperature, 275 °C; sheath gas flow, 30 arbitrary units; auxiliary gas flow, 3 arbitrary units; capillary voltage, 38 V; ESI spray voltage, 4.5 kV. The target was fixed at 1 × 107 ions and the automatic gain control was turned on. The instrument was operated in the data-dependent acquisition mode, allowing the automatic switching between MS and MS/MS. The MS survey scan was performed from m/z 300-2000 in the Orbitrap, using a resolution set at 30,000 (at m/z 400), and the ion population was held at 5 × 105 through the use of automatic gain control. The five most abundant ions (threshold 500 counts, charge states higher than +1) were further selected for collision induced dissociation (CID) experiments. The CID fragment ions were detected in the linear ion trap. A normalized collision energy of 35% was used, with an activation q of 0.25 and an activation time of 30 ms. For tandem mass spectrometry in the linear ion trap, the ion population was set to 1 × 104 and the precursor isolation width was set to 3 m/z units. Detection was also performed in the Orbitrap at a target resolution of 15,000 at m/z 400, whereas the ion population was set at 2 × 105, and the precursor isolation width to 3 m/z units to separate experiments. FTMS/MS data were collected in profile mode and ion trap data in centroid mode. Relative quantification of histone modifications was determined as described before,25 by measuring the area of the extracted ion chromatogram peak corresponding to a specific modified peptide normalized to the sum of the peak areas corresponding to all observed modified forms of this peptide.

Data analysis was performed both manually and using the Sequest algorithm included in the Bioworks Browser (Version 3.3.1 SP1, Thermo, San Jose, CA). In the latter case, MS and MS/MS spectra were searched against a restricted database consisting of human histone sequences. Regarding propionylated peptides, the criteria previously described by PlazasMayorca et al. were used.15 Briefly, propionylation was defined as a fixed modification on the N-terminus of the peptides (+56.026 Da), whereas methionine oxidation (+15.995 Da) was considered as a variable modification. Other variable posttranslational modifications of lysine residues were defined as follows: propionylation (+56.026 Da), acetylation (+42.011 Da), mono- (+70.042 Da), di- (+28.031 Da), and trimethylation (+42.047 Da). Monomethylation (+70.042 Da) was set as the sum of propionylation (+56.026 Da) and monomethylation (+14.016 Da) because monomethylated residues can still be propionylated.

Results and Discussion We developed a fast and reproducible method involving UHPLC coupled to a high resolution/high mass accuracy LTQOrbitrap mass spectrometer to characterize core histone variants/modifications. First, the intrinsic characteristics of the approach for histone profiling and identification were evaluated. Artifactual oxidation potentially occurring during sample preparation as well as associated chromatographic changes were then assessed. Method reproducibility was also thoroughly determined by analyzing analytical and biological replicates. Last, histone tryptic digests were analyzed by LC-MS/MS to further confirm the nature and relative abundances of histone PTMs. UHPLC-MS Analysis of Unfractionated Intact Histones as a Fast Screening Tool. RP-HPLC can be regarded as a method of choice for efficiently purifying individual histones from acid-extracts of chromatin.23 The best chromatographic separations are commonly obtained with trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) as mobile phase additives.27 Under classical RP-HPLC conditions, other ionpairing agents such as formic acid often cause peak broadening, thus leading to poorer chromatographic resolution. However, regarding LC-MS coupling, significant ion suppression can be observed when using TFA or HFBA, which can result in a ∼10 fold lower signal for given histones when comparing mobile phases with 0.06% TFA or 0.04% HFBA to those containing 0.4% formic acid.27 Moreover, in certain instances, TFA adducts can also be observed on histone proteins, thus leading to more complex and difficult mass spectra to interpret.13,19 Signal suppression effects and formation of TFA adducts can be somewhat minimized by either lowering the percentage of TFA in the mobile phases or by optimizing the source parameters.13,19 Since the intensity of TFA adducts can somehow fluctuate from one LC-MS system to the other, Su et al. recently mentioned that they are moving from their LC-MS method that used TFA to a TFA-free approach for robust comparisons across LC-MS platforms.13 Nevertheless, TFA is still widely used for LC-MS analysis of histone proteins. Under common conditions, the average runtime is >60 min.13,19,21 In this context, we have developed an UHPLC-MS method using formic acid as mobile phase modifier, which enables a fast and efficient separation of unfractionated acid-extracted histone proteins and analysis of variants and modified forms in less than 20 min (Figure 1A). The chromatographic system was interfaced to a high resolution/high mass accuracy LTQJournal of Proteome Research • Vol. 9, No. 10, 2010 5503

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Figure 1. Online UHPLC-MS analysis of unfractionated acid-extracted histones from WI-38 primary human fibroblasts. (A) Total ion chromatogram. (B and C) Mass spectra of histones H4 and H3.1, respectively. (Insets) Corresponding deconvoluted monoisotopic spectra of histones H4 and H3.1.

Orbitrap instrument. Under these conditions, the core histones H2A, H2B, H3, and H4 are all eluted and resolved within 2 min (Figure 1A and Figures S1A and S1C, Supporting Information). This kind of fast chromatographic separation is slighlty less efficient in terms of resolved peaks than an optimized 1.5 h-method27 but gives similar or even better results than a previously reported 40 min-routine method for high-throughput analysis.20 1. Identification of Intact Histones. Identification of intact histone species was first realized by matching experimentally 5504

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determined masses with theoretical ones, obtained from known amino acid sequences and the corresponding expected PTMs (Table 1). Thanks to the high resolution and the high mass accuracy of the Orbitrap analyzer, an average mass accuracy of ∼6 ppm was obtained for all the core histones detected, as determined from deconvoluted monoisotopic masses and after external calibration of the instrument (Table 1 and Figure 1B-C). For instance, masses of H4 (considering N-terminal acetylation and 2 methyl groups) and H3.1 (considering 4 methyl equivalents) can be measured with mass accuracies of 5.3 and 10.4 ppm,

technical notes

Fast Profiling of Histone Post-Translational Modifications a

Table 1. Masses and Accuracies of the Main Histone Species Observed by UHPLC-MS histones b

H4 H2B.Ac H2A.Z H2A.Oc H2A.C,D,I,N,Pc H3.2b H2A.X H3.1b

accession #

PTMs

theoretical masses (Da)

observed masses (Da)

∆m

error (ppm)

P62805 P62807 P0C0S5 Q6FI13 P0C0S8 Q71DI3 P16104 P68431

N-terminal acetylation + dimethylation None None N-terminal acetylation N-terminal acetylation 4 methyl equivalents N-terminal acetylation 4 methyl equivalents

11,299.38 13,766.52 13,413.51 13,997.86 13,993.92 15,303.53 15,046.39 15,319.50

11,299.44 13,766.65 13,413.56 13,997.92 13,993.99 15,303.61 15,046.48 15,319.66

0.06 0.13 0.05 0.06 0.07 0.08 0.09 0.16

5.31 9.44 3.73 4.29 5.00 5.23 5.98 10.44

a Theoretical and observed masses are monoisotopic masses. Theoretical masses were calculated based on known primary sequences and PTMs. abundant form. c Most abundant variant in the peak labeled H2B, H2A1, H2A2 presented in Figure 1.

respectively (Table 1). These values are in excellent agreement with the ones of 9 ppm (for H2A)13 and 5 ppm (for H4)19 previously obtained on Orbitrap and FT-ICR instruments, respectively. Such mass accuracy corresponds to a ∼0.1 Da error in mass, which is ∼10 fold better than the average ∼1 Da mass error previously reported for Q-TOF instruments.13,27 One of the strengths of such protein profiling approach is the potential comprehensive characterization of abundant PTMs and variants. For instance, acetylation and methylation of histones H4 and H3.1 can be easily evidenced, respectively (Figure 1B-C). Moreover, mass differences arising from those modifications were measured with high precision, 42.00 and 14.01 Da on average for H4 acetylation and H3.1 methylation, respectively (Figure 1B-C). However, up to (0.05 Da fluctuations could be observed for these values. Thus, potential distinction of modifications with the same nominal mass, such as acetylation and trimethylation (42.01 and 42.05 Da, respectively) can not be strictly envisioned at the intact protein level. These results are in good agreement with those of Thomas et al., who studied histone H3 PTMs using an FT-ICR instrument.28 Histone identity was further confirmed by performing topdown UHPLC-MS/MS experiments. The CID fragmentation of precursor ions at m/z 700 and m/z 1150 was performed in the LTQ with an isolation width of 70 mass units. These different parameters were optimized by injecting pure histone samples (data not shown). Similarly, Bondarenko et al. recently reported the top-down LC-MS/MS analysis of heavy and light chains from a monoclonal antibody on an LTQ-Orbitrap instrument, through the CID fragmentation of a single precursor ion at m/z 1000 by isolating an m/z 100 range.29 Since core histones exhibit distinct charge state distributions, the selection of two different precursor ions with a m/z 70 isolation window was required to efficiently and automatically fragment all core histone species in a single experiment. Figure 2 shows the LC-MS/MS spectra of two histones, H4 and H3.1, that exhibit different levels of PTM complexity. Up to 15 distinct fragment ions can be observed for these proteins with an average mass accuracy better than 2.5 ppm (Figure 2). Although yielding enough data to unambiguously identify the protein, CID fragmentation of intact histones does not enable as complete PTM characterization as electron capture dissociation (ECD) fragmentation with potential single residue resolution. Nevertheless, useful information regarding PTM localization can still be obtained. For instance, the presence of both H4 unacetylated (i.e., only acetylated on the N-terminal residue) and monoacetylated b233+ fragment ion (at m/z 804.827 and m/z 818.833, respectively) highlights the acetylation of one lysine residue among Lys5, Lys8, Lys12 and Lys16 (Figure 2A). Similarly, the presence of the y405+ fragment ion in the MS/MS spectrum of H3.1 (Figure 2B) confirms the presence of a cysteine residue

b

Most

in position 96, an amino acid residue distinguishing H3.1 from H3.2 and H3.3 species. Lastly, it should be mentioned that similar MS/MS spectra were obtained when the corresponding HPLC-purified histones were analyzed, thus demonstrating the efficiency of this online automatic top-down UHPLC-MS/MS approach (data not shown). 2. Evaluation of Potential Artifacts Linked to Partial Histone Oxidation during Sample Preparation. Partial artifactual oxidation of certain methionine and/or cysteine residues from histones may happen during sample preparation (e.g., acid-extraction, HPLC-fractionation).24 Partial oxidation of histones can drastically complicate MS analysis because the resulting signals may decrease the global detection sensitivity and also alter the relative abundances of histone PTMs. For instance, this may be due to isotopic overlaps of monomethylated (+14.02 Da) and mono-oxidized (+15.99 Da) forms. To overcome this drawback, Pesavento et al. used a mild performic acid oxidation to completely oxidize methionine and cysteine residues without modifying other amino acids or the levels of PTMs on histones.24 Thus, to evaluate whether or not our sample preparation may induce artifactual histone oxidation, unfractionated acidextracted histones were oxidized under the conditions described by Pesavento et al.,24 and the resulting PTM profiles compared with those previously obtained with untreated histones. No significant differences were observed for all histone species, as shown in Figure 3 for H4, H2B and H3.1. This indicates that there is no major interference due to oxidation during our sample preparation prior to UHPLC-MS analysis (in fact limited to acid-extraction). Nevertheless, it should be mentioned that significant oxidation levels were detected when HPLC-purified histones were submitted to UHPLC-MS analysis (data not shown). Altogether these data suggest that, under our conditions, mild performic acid oxidation of unfractionated histones is not mandatory prior to UHPLC-MS analysis. However, in certain instances, the particular chromatographic separation of oxidized histones24 can be of special interest to obtain better chromatographic resolution between given species (Figure 3 and Figures S1B and S1D, Supporting Information). Nevertheless, it should be mentioned that a potential limitation of such an approach is that information regarding oxidation occurring in vivo is lost.30 3. Evaluation of the Reproducibility of the UHPLC-MS Method. In order to compare PTM and/or variant profiles of given histone species observed under different conditions (e.g., following administration of a given drug), validated approaches are required. Thus, it becomes mandatory to assess their reproducibility, which represents a critical component of labelfree methodologies. A few parameters can influence the Journal of Proteome Research • Vol. 9, No. 10, 2010 5505

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Figure 2. Online top-down UHPLC-MS/MS analysis. (A) Histone H4 and (B) Histone H3.1. Corresponding fragment coverage maps are also shown.

robustness of LC-MS profiles, such as sample preparation, instrument and biological variations. In this context, we evaluated both the instrumental reproducibility through the duplicate injection of three different 5506

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samples on three distinct days and the biological variation combined with sample preparation variation by analyzing three independent biological replicates (Table 2). Relative abundances of histone modified forms/variants (present at >5%

Fast Profiling of Histone Post-Translational Modifications

technical notes

Figure 3. Online UHPLC-MS analysis of unfractionated acid-extracted histones after mild performic acid oxidation. (A) Total ion chromatogram. (B) Deconvoluted mass spectra of untreated versus oxidized histones H4, H2B and H3.1. Indicated mass shifts correspond to one oxidized methionine residue (+∼32 Da) for H4, two oxidized methionines (+∼64 Da) for H2B, and two oxidized methionines and two oxidized cysteine residues (+∼160 Da) for H3.1. Table 2. Evaluation of UHPLC-MS Reproducibilitya histones

analytical CVb (n ) 3)

biological CVc (n ) 3)

H4 H2B H2A1 H2A2 H3.2 H3.1

3.6% 6.4% 7.9% 10.5% 12.0% 8.2%

14.2% 6.3% 16.3% 15.9% 21.2% 20.4%

a CVs were calculated on histone modified forms/variants with relative abundances >5% in each chromatographic peak. b Analytical CVs were calculated from three independent samples injected in duplicate experiments performed on three distinct days. c Biological CVs were calculated on three independent biological samples analyzed on three distinct days.

relative abundance) were determined for the different samples and coefficients of variation (CVs) were calculated. Analytical replicates yielded CVs ranging from 4 to 12% (∼8% on average), while biological replicates showed CVs between 6 and 21%

(∼16% on average) for several different histone species (Table 2). Thus, UHPLC-MS data yielded similar results to previously reported ones by LC-MS with CVs of less than 5% for abundant histone H2A variants obtained from five distinct preparations of a bovine histone standard13 and less than 10% for H2B variants with >10% relative abundance in global chromatin (three repeated samplings of untreated cells).19 In addition, the reproducibility of the separation itself was found to be excellent, with retention times not varying more than (0.1 min. Thus, the developed UHPLC-MS methodology was demonstrated to be efficient and reproducible for assessing the global combinatorial modification profiles of core histones and variants in a high-throughput manner. As mentioned above, top-down MS/MS analysis (using CID fragmentation) of intact histones did not enable characterization of PTMs. Thus, to further confirm PTMs and isoform identities as well as their Journal of Proteome Research • Vol. 9, No. 10, 2010 5507

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relative distributions, histones were digested with trypsin and analyzed by LC-MS/MS. LC-MS/MS Analysis of Histone Tryptic Digests. While LC-MS-based profiling approaches can rapidly highlight potential histone PTMs or variants of interest, subsequent experiments are further needed to properly identify and characterize the species in question. The classical workflow for postprofiling characterization of histone forms commonly encompasses LC-MS(/MS) analysis of histone tryptic digests after chemical modification. For instance, histone propionylation combined with trypsin digestion constitutes a very convenient way to determine histone PTM localization and can also be used to compare protein populations between different samples (labelfree relative quantification).31,32 Thus, in an attempt to mimic the postprofiling workflow, unfractionated histone acid-extracts were treated with propionic anhydride prior to trypsin digestion as recently described,15 and the semiquantitative results obtained on three independent biological replicates were further compared to those from the UHPLC-MS method on the same samples. Relatively simple examples, dealing with H4 and H2A.Z acetylation and H2A.O/Q variants, are given below to illustrate the results. Figures 4A-B shows an integrated picture of measurements made either at the protein or at the peptide level, regarding acetylation of histones H4 and H2A.Z. For these two proteins, histone-specific tryptic peptides bearing the main modification sites were identified and monitored. LC-MS/MS data further confirmed that H4 and H2A.Z were acetylated on N-terminal peptides (Gly4-Arg17) and (Ala1-Arg19), respectively. As an example, Figure S2 depicts the MS/MS spectrum of histone H4 peptide (Gly4-Arg17) acetylated on Lys16. As may be seen at first glance on Figure 4A-B, similar quantitative data were obtained with proteinand peptide-based approaches, with ∼55-60% of histone H4 being unacetylated, ∼30-35% monoacetylated, and ∼5-10% diacetylated. These values are in very good agreement with data previously obtained from WI-38 normal human diploid fibroblasts.33 Similar results were also obtained on H2A.Z with ∼85-90% being unacetylated and the remaining monoacetylated. These results further confirm the efficiency of UHPLC-MS for the fast and accurate profiling of histone PTMs. Although more efficient for obtaining deeper structural insights on histones (e.g., modification sites), bottom-up approaches can lead to peptides shared by distinct protein variants, thus not providing variant-specific information. This is especially true when enzymatic digestion experiments are performed on unfractionated digests. For instance, Figure 4C gives the example of H2A.O and H2A.Q species, which can not even be separated by off-line HPLC purification. In this particular case, enzymatic digestion always leads to overlapping data, while only intact protein mass measurement can deliver variant-specific information. Thus, slight but significant acetylation differences (∼5-10%) between histones H2A.O and H2A.Q can be obtained by UHPLC-MS, with almost no acetylation observed on intact H2A.Q (Figure 4C). Through these examples, we demonstrate that our UHPLC-MS method rapidly provides convincing semiquantitative data on major histone PTMs and variants in complex biological samples. The identity and relative distributions of histone isoforms were further confirmed by a more conventional bottom-up approach. Extensively modified histones such as H3 variants can easily be analyzed by our UHPLC-MS approach (Figure 1C). However, in that case and after trypsin digestion, PTMs are spread over numerous tryptic peptides, so that combinatorial information is 5508

Journal of Proteome Research • Vol. 9, No. 10, 2010

Figure 4. Relative isoform populations deduced from measurements performed either at the protein or at the tryptic peptide level for histones (A) H4, (B) H2A.Z, and (C) H2A.O. Experiments were performed on three independent biological replicates.

lost, which renders comparison of protein- and peptide-based approaches difficult. To encompass this limitation, it would be possible to work on the 50 N-terminal residues obtained upon

technical notes

Fast Profiling of Histone Post-Translational Modifications Glu-C digestion of histone H3 (middle-down MS) that can be efficiently fragmented using ECD to generate site-specific information.34

Conclusions In this work, we report the development of a fast, efficient and reliable top-down UHPLC-MS method for profiling the main histone modifications and variants. We demonstrate that our method enables in less than 20 min the separation and identification of histone PTMs and variants. The approach was demonstrated to be highly reproducible through the analysis of both analytical and biological replicates. With a sample preparation limited to histone extraction, the occurrence of process-induced modifications (such as oxidation) is also minimized. Detected isoforms can also be semiquantitatively measured through a labelfree approach and give similar results than those obtained on tryptic peptides. In conclusion, our UHPLC-MS approach is a significant improvement to existing methods for analyzing histones whenever multiple samples need to be analyzed.

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