Gene-Specific Characterization of Human Histone H2B by Electron

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Gene-Specific Characterization of Human Histone H2B by Electron Capture Dissociation Nertila Siuti,† Michael J. Roth,† Craig A. Mizzen,§,| Neil L. Kelleher,*,†,‡,|,⊥ and James J. Pesavento‡ Department of Chemistry, Center for Biophysics and Computational Biology, Department of Cell and Developmental Biology, Institute for Genomic Biology (IGB), and The Center for Top Down Proteomics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Received August 16, 2005

The basis set of protein forms expressed by human cells from the H2B gene family was determined by Top Down Mass Spectrometry. Using Electron Capture Dissociation for MS/MS of H2B isoforms, direct evidence for the expression of unmodified H2B.Q, H2B.A, H2B.K/T, H2B.J, H2B.E, H2B.B, H2B.F, and monoacetylated H2B.A was obtained from asynchronous HeLa cells. H2B.A was the most abundant form, with the overall expression profile not changing significantly in cells arrested in mitosis by colchicine or during mid-S, mid-G2, G2/M, and mid-G1 phases of the cell cycle. Modest hyperacetylation of H2B family members was observed after sodium butyrate treatment. Keywords: histone • chromatin • post-translational modifications • top down mass spectrometry • electron capture dissociation

Introduction The regulation of chromatin is achieved through a variety of coordinated pathways involving interactions between macromolecular complexes with DNA and DNA-associated proteins. Histones, the main structural proteins associated with DNA, are often modified which in turn affects chromatin organization as well as gene expression.1 Such modifications include acetylation, methylation, phosphorylation and ubiquitylation, which may occur individually or in combination.2 The different post-translational modifications (PTMs) can modulate histone:DNA interactions and create new sites of dynamic interaction for protein complexes responsible for regulating chromatin compaction and DNA-templated activities including transcription, replication and repair.3,4 This introduces a new dimension of regulation beyond that encoded in the DNA sequence.5,6 Another mechanism of regulation which functions in concert with histone modification is the differential expression of histone variants.7,8 Relative to the wealth of information available on various PTMs, the significance of the amino acid substitutions that distinguish these related gene products is poorly understood, with little or no information available on how they affect nucleosome and chromatin dynamics. The H2A and H2B protein families are far more heterogeneous than H39 and H4 in humans (15 different gene sequences encode the * To whom correspondence should be addressed. 600 S. Mathews, 53 Roger Adams Laboratory, Urbana, IL 61801. Tel: (217) 244-3927. E-mail: [email protected]. † Department of Chemistry. ‡ Center for Biophysics and Computational Biology. § Department of Cell and Developmental Biology. | Institute for Genomic Biology (IGB). ⊥ The Center for Top Down Proteomics. 10.1021/pr050268v CCC: $33.50

 2006 American Chemical Society

same protein sequence in the case of H4).10 Seventeen H2B genes are known in the human genome, two of which are believed to be pseudogenes and fifteen that encode eleven different H2B gene products.11 These variants may have acquired functional differences in metazoans since only two H2B gene family members (H2B.1 and H2B.2) are known in lower Eukarya such as Tetrahymena thermophila and Saccharomyces cerevisiae.12,13 Studies of H2B modification in yeast have shown that ubiquitylation at lysine 123 (K123) is required for histone H3 methylation at K4 and K79.14,15 However, other work suggests that ubiquitylation of H2B is dispensable for monomethylation of H3 at K4 and K79, but is necessary for processive methylation to occur.16 Most recently, it was reported that S10 of H2B in yeast (S14 in humans) is phosphorylated during apoptosis induced by hydrogen peroxide.17 Work on Tetrahymena H2B shows evidence for heterogeneity of methylation states at the N-terminal alanine which includes mono, dimethylation and trimethylation.12 Furthermore, N-terminal peptides resulting from proteolytic digests revealed K3 and K4 to be modified (acetylated and trimethylated).12 Peptide mass fingerprinting of bovine H2B is consistent with acetylation or monomethylation of K5 and acetylation of K12, 15, and 20.18 K43 was suggested to be monomethylated and K85 acetylated. Analyses of data from the mouse and human genome sequencing projects have revealed that 10 or more genes encoding nonallelic variants of H2A and H2B exist in these species.19 Assessing the relative expression of these variants at the level of mRNA is problematic because of the high degree of DNA sequence conservation between the genes of these variants. Similarly, the high degree of amino acid sequence conservation poses challenges to assessing their expression at the protein level using Bottom Up Mass Spectrometry.20 Recent Journal of Proteome Research 2006, 5, 233-239

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research articles developments in the field of mass spectrometry (MS), including analysis of intact proteins, have facilitated the identification of different histone isoforms and characterization of their PTMs.21 Recently, Top Down MS with Electron Capture Dissociation (ECD) of several H2B forms simultaneously enabled the identification and localization of modifications and identification of gene family members present in the mixture from Tetrahymena.12 However, this approach is generally unable to assign PTMs to individual sequence variants or differentially modified forms present in the original mixture. PTM identification on a gene-specific basis by tandem MS (MS/MS) after careful isolation of intact histone variants has yet to be reported. Here, we combine a Quadrupole-FTMS hybrid (Q-FTMS)22 with Top Down fragmentation of specific isotopic distributions to describe the most abundant forms of H2B expressed in asynchronously growing HeLa cells, and in cells in different portions of the cell cycle in synchronized cultures, in cells arrested in mitosis by colchicine, and in cells treated with the histone deacetylase inhibitor sodium butyrate. Measurement of protein ion relative ratios and fragment ion relative ratios following high-resolution isolation and ECD fragmentation resulted in gene-specific identification of seven H2B gene products and one monoacetylated form. H2B.A was the most abundant form and was found to be ∼20% acetylated in asynchronous cell populations. Butyrate treatment resulted in a modest hyperacetylation of several H2B variants. In contrast, the mass spectrum of H2B from cells treated with colchicine was remarkably similar to that from asynchronous HeLa. We also did not find evidence of differential expression of H2B variants during the cell cycle.

Materials and Methods Purification of Histone H2B from HeLa S3 Cells. HeLa S3 cells were maintained at a density of 2-3 × 105 cells/mL in spinner flasks in Joklik’s modified MEM supplemented with 5-10% newborn calf serum. Cells were collected by centrifugation, washed twice in cold tris-buffered saline (TBS) and then lysed in nucleus isolation buffer (15 mM Tris-HCl pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 250 mM sucrose, 1 mM dithiothreitol, 5 nM microcystin-LR, 500 µM 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 mM sodium butyrate), supplemented with 0.3% NP-40 followed by low-speed centrifugation to prepare nuclei as described previously.23 Histones were extracted by incubating isolated nuclei with 0.4 N H2SO4 on ice for 2-4 h with intermittent mixing. Acid insoluble material was removed by centrifugation, trichloroacetic acid was added to the supernatant (20% w/v final) and the precipitated core histones recovered by centrifugation after several hours of incubation on ice. The pellet was washed with acetone +0.1% HCl, followed by two washes with 100% acetone, dried, resuspended in water and then oxidized by treatment with performic acid.24 H2B was then separated from other proteins by reverse phase HPLC (RPLC) on a Vydac C18 column (4.6 × 250 mm, Separations Group, Hesperia, CA). Approximately 100 µg of crude acid-extracted protein was separated using a multistep gradient from 100% A (5% acetonitrile, 0.1% TFA) to 100% B (90% acetonitrile, 0.094% TFA) at a constant flow rate of 0.8 mL/min. Protein elution was monitored at 214 nm. H2B (approximately 25 µg) eluted as a single peak at ∼43% buffer B. Identity and purity were assessed by electrophoresis in polyacrylamide gels containing SDS. Metabolic Labeling of Cells with L-Methionine-methyl-13Cmethyl-d3. HeLa cells were grown in Joklik’s media prepared 234

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Figure 1. Broadband spectrum of histone H2B from asynchronously growing HeLa S3 cells. (A) Spectrum of intact H2B gene products after reverse phase HPLC purification. The oxidized, monoisotopic mass is listed above each molecular species. (B) ECD MS/MS spectrum (132 scans) of peak 1 (13830.7 Da) from Figure 1A with representative fragment ions and SWIFT isolated parent ion shown as insets on the left and right, respectively.

without methionine, supplemented with 5% dialyzed newborn calf serum (NCS) and 0.2 mM L-methionine-methyl-13C-methyld3 (Isotec, West Chester, OH). Cells were cultured for 168 h to ensure that the intracellular pools of methionine, S-adenosylmethionine (methyl donor for protein methylation), and the H2B proteins themselves, were saturated with labeled methionine. H2B was prepared by isolating nuclei and RPLC purification of acid extracts as described above. Cell Cycle Synchronization and Perturbation. Asynchronous cultures were treated with media containing 10 mM sodium butyrate or 1 µM colchicine for exactly 24 h. Nuclei were isolated and histones prepared by acid extraction as described above. Cultures at a density of 3 × 105/mL in Joklik’s modified MEM containing 10% NCS were synchronized by a double thymidine block procedure.25 Mid-S, mid-G2, M/G2, and mid-G1 samples were collected at 4, 8, 10.5, and 15 h postrelease, respectively, based on hourly measurements of DNA content by flow cytometry. Cells harvested at these time points were collected by centrifugation, washed twice in ice cold TBS and then either snap frozen in liquid nitrogen or processed for nuclear isolation directly. Mass Spectral Analysis by ESI/Q-FTMS. Data were acquired on a custom 8.5 T22 or 9.4 T26 (ECD of peak 5 and peak 3 in Figure 1A) Quadrupole-FT ICR MS with an ESI source operated in positive ion mode. Sample was directly infused into a fused silica ESI emitter with a 50 µm i.d. tip (New Objective, Woburn, MA) at a flow rate of 300 nL/min. The capillary inlet was heated by applying 3.5 A and the capillary voltage was set typically at 2150 V. Alternatively, the NanoMate 100 (Advion BioSciences, Ithaca, NY) used 15 µL of solution from each well to automatically establish the spray. Typically, 15 µL samples were enough for more than 100 min. of stable nanospray, providing ample time to acquire high-quality MS and MS/MS scans of four to eight isotopic distributions per sample.

research articles

Gene-Specific Characterization of Human Histone H2B

Figure 2. Characterization of a mixture of isobaric species (H2B.Q and H2B.E). (A) ECD (51 scans) of peak 2 (13844.6 Da) from Figure 1A. (B) Key fragment ions in the 625-725 m/z region reporting on the presence of H2B.Q and H2B. E. (C) Sequence alignment of H2B.E and H2B.Q forms. Amino acid sequence difference are underlined.

Quadrupole Enhancement. A quadrupole (ABB Extrel, Houston, TX) was used to select the 17+ and 15+ charge state of histone H2B species, which were then accumulated in a rf only octupole equipped with a dc voltage gradient for improved ion extraction. The quadrupole window was set at ∼40 m/z and centered around the H2B peak of interest. Stored Waveform Inverse Fourier Transform (SWIFT)27 was used to further filter the selectively enhanced charge states and the data was collected using MIDAS.26,27 Electron Capture Dissociation. ECD was performed by applying 5 A through a dispensor cathode filament (Heatwave Technologies, Crescent Valley, BC). During the ECD event, ∼10 V was applied on the grid potential while ∼9 V were sent through the filament for optimal ECD.28 Typically, 200 cycles of ECD were performed, with individual irradiation times of 3 ms and a 10 ms relaxation time between each cycle. ECD MS/ MS spectra were internally calibrated using 3-4 identified z• ions from the unmodified C-terminal region. Infrared Multiphoton Dissociation (IRMPD). Following quadrupole and SWIFT based ion isolation, irradiation using an IR beam from a 75 W (10.6 µm) CO2 laser (Synrad Inc., Mukilteo, WA) was performed for 150 ms at ∼50% power.29 Software and Databases. The gene-specific web server used for identification/characterization had a 1.6 GHz dual processor Athlon 2200 + CPU. ProSight PTM, a web-based software and database suite https://prosightptm.scs.uiuc.edu30 was used to query a custom human database created by shotgun annotation.21 Gene-specific identifications were based on matching multiple fragment ions that distinguished one specific H2B form from its closest relative in the gene family.

Figure 3. Isolation and identification of two forms of H2B (H2B.J and H2B.K/T). (A) ECD MS/MS (147 scans) with c24 fragment ion from H2B.K/T and -14.05 Da satellite peak from H2B.J. (B) IRMPD (225 scans) with representative fragment ions, H2B.J y57 and the -16.01Da satellite peak corresponding to H2B.K/T.

Results and Discussion An ESI/Q-FTMS spectrum of RPLC-purified histone H2B from asynchronously growing HeLa cells revealed five main isotopic distributions g3% relative abundance (Figure 1A). As noted by other researchers, partial oxidation of methionines and cysteines resulting from procedural and instrumental sources can create complex spectra during Top Down MS/MS.31 Therefore, we intentionally oxidized the two methionines in H2B with performic acid prior to MS, resulting in >99% conversion to methionine sulfone (data not shown). The molecular species observed in Figure 1A are very close in mass (separated by 14-16 Da) suggesting the possibility that this population contained multiple variants of H2B, forms bearing multiple methylations,31 or both, as is the case for H332 and H4.21 To separate the potential overlap of methylated H2B (∆m ) +14.016 Da) with an isobaric amino acid variant (i.e., Asp to Glu, ∆m ) +14.016 Da), cells were labeled with L-methionine13 C-methyl-d3, to increase the mass of H2B by 4 Da for each labeled Met incorporated or for each labeled methyl group transferred by histone methyltransferases. As shown in Figure 7 (compare panels A and C), the MS profile for the labeled and unlabeled H2B was nearly identical, except that the labeled H2B was shifted by the addition of 8 Da (all known H2B variants have two methionines, 2 × 4 Da ) 8 Da shift (Figure 7C). The isotopic distribution for each labeled H2B molecular form was carefully matched with theoretical distributions. Broadening due to methylation or incorporation of nonlabeled methionine was not apparent. Furthermore, the mass difference between the labeled molecular species was the same as the mass difference for the unlabeled species. Taken together, this suggests that much of the heterogeneity in the MS profile of H2B is due to amino acid sequence variation rather than methylation or other PTMs, although these are potentially present in minor abundance below our detection limit. Journal of Proteome Research • Vol. 5, No. 2, 2006 235

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Figure 4. MS/MS of peak 4 (13860.4 Da) (H2B.B) from Figure 1A. (A) ECD (75 scans) of peak 4 with SWIFT isolated parent ion and H2B.B reporting fragment ion c42. (B) ProSight PTM output for H2B.B.

Figure 5. Characterization of mono-acetylated H2B.A from asynchronous HeLa. (A) ECD MS/MS spectrum (63 scans) of peak 5 (13873.8 Da) from Figure 1A. (B) Broadband spectrum of histone H2B from asynchronous HeLa cells. (C) The c25 fragment ion reporting on acetylated-H2B. A.

To confirm this observation, each of the 5 major molecular species in both labeled and unlabeled H2B mass spectra were carefully isolated by SWIFT and analyzed by MS/MS using ECD fragmentation. The isolated precursor ion and MS/MS spectrum of the most abundant form (peak 1, Mr of 13 830.7 Da) is shown in Figure 1B. The calibrated fragment ion masses were used to probe the entire ProSight database using a 25 ppm tolerance. The top hit was the H2B.A isoform. Of the 58 observed fragment ions, 18 and 15 match c and z• ions, respectively, with an overall mass difference (∆m) of -0.19 Da for the intact protein. IRMPD was also performed on this species and the resulting b and y ions confirmed the presence of H2B.A (Supporting Information Figure 1A). R-acetylation at the N-terminus, commonly found on histone H2A and H4, was not observed in H2B.A or any of the other H2B forms present, consistent with past analyses of bulk histone H2B.33,34 The isotopic distribution of the second most abundant component in Figure 1A (peak 2, Mr of 13 844.6 Da) was isolated and subjected to ECD (Figure 2A). Of the 158 fragment ions generated, 41 c and 38 z• ions matched the H2B.Q isoform. H2B.E has the same mass as H2B.Q (13 844.5 Da) but with three amino acid changes: Glu 2 Asp (∆m ) -14 Da), Ala 21 Val 236

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Figure 6. Comparison of broadband profiles of asynchronous H2B and H2B during the cell cycle (time points from mid-S, midG2, G2/M, and mid-G1). (A) Broadband spectrum of H2B from asynchronous HeLa and corresponding flow cytometry analysis of cellular DNA content (inset). (B) Broadband spectrum of H2B from mid-S (4 h) time point. (C) Broadband spectrum of H2B from mid-G2 (8 h) time point. (D) Broadband spectrum of H2B from G2/M (10.5 h) phase. (E) Broadband spectrum of H2B from midG1 (15 h) time point. Analysis of cellular DNA content by flow cytometry is shown for each time point (inset).

(∆m ) +28 Da), and Ile 39 Val (∆m ) -14 Da, Figure 2C). The presence of -14 Da satellite peaks for c15 and c19 and +14 Da satellite peaks for c25, c26 and c32 confirmed that both H2B.Q and H2B.E were present in peak 2 (Figure 2B). The presence of only a single c39 fragment ion and z• type ions below z86 (11 such ions) is entirely consistent with isobaric fragment ions from this mixture of H2B.Q and H2B.E. In addition to identifying unmodified variants, these data also suggest the possible presence of monomethylation somewhere in the N-terminal domain of H2B.A (also Mr of 13844.6). MS/MS of H2B from asynchronous HeLa cells metabolically labeled with L-methionine-methyl-13C-methyl-d3 (Figure 7C) was performed to explore the possible presence of monomethylated H2B.A. However, satellite peaks of +18 Da representing a 4 Da augmentation of the 14 Da shift normally associated with protein methylation were not observed from the analysis of the MS/MS data of peak labeled 2′ in Figure 7C. This precludes the presence of monomethylated H2B.A in this sample with a detection limit of ∼5% relative abundance. On the basis of the observed fragment ion relative ratios35 from different charge states we conclude that peak 2 contained ∼60% H2B.Q and ∼40% H2B.E.

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Gene-Specific Characterization of Human Histone H2B

Figure 7. Profile of H2B from HeLa cells treated with colchicine, or sodium butyrate or after metabolic labeling with L-methioninemethyl-13C-methyl-d3. (A) Broadband of H2B from asynchronous HeLa cells. (B) Broad band of H2B from cells following overnight treatment with 1 µM colchicine. (C) Broad band spectrum of H2B from HeLa cells grown in media containing 0.20 mM L-methionine-methyl-13C-methyl-d3. (D) Broadband spectrum of H2B following 24 h treatment with 10 mM sodium butyrate and a ECD fragment ion helping to localize monoacetylation of H2B.A at lysine 15 (inset).

ECD of the species with the lowest Mr value (13 814.8 Da) in the spectrum of Figure 1A (peak 3) generated 75 fragment ions. Twenty-five match c and nine match z• ions indicating the presence of H2B.K (13 814.5 Da) or H2B.T (13 814.5 Da) and H2B.J (13 816.5 Da) as the major forms present. H2B.K and H2B.T have exactly the same sequence and are encoded by genes on chromosome 6. We refer to this species as H2B.K/T. H2B.J and H2B.K/T differ from each other by two amino acids (positions 2 and 124). Compared to the H2B.J sequence, z• ions showed a -16 Da mass discrepancy due to a serine to alanine variation at position 124 in the C-terminus of H2B.K. Also, ECD performed on a 9.4 T ESI Q-FTMS showed 9 c ions having -14.05 Da satellite peaks out of a total of 18 c ions confirming the presence of H2B.J (consistent with E to D substitution; Figure 3A). IRMPD generated y ions which confirmed the presence of H2B.K/T and H2B.J (Figure 3B and Supplementary

Figure 1B); no b ions from H2B.J were detected in the IRMPD experiment. As shown in Figure 3 (and Supporting Information Figure 1B), H2B.J is present at ∼30% and H2B.K/T is present at ∼70% as determined by the fragment ion relative ratios.35 Another form of H2B with a Mr value of 13 860.4 Da was SWIFT isolated (peak 4 in Figure 1A) and dissociated by ECD. This allowed identification of this form as H2B.B based on matching 29 c ions and 36 z• ions from a total of 106 fragment ions (Figure 4A,B). Relative to H2B.A this form had an alanine to a threonine substitution (∆m ) 30.0 Da) at the fourth residue. Comparison to the H2B.A sequence revealed that the c ions displayed a +30.02 Da shift due to the presence of threonine at position 4. On the basis of Mr values alone, peak 5 in Figure 1A and Figure 5B (Mr value of 13 873.8 Da) could consist of acetylated H2B.A (13872.5 Da) and/or H2B.F (13 874.5 Da). Fragment ion c25 had a mass shift of +42.01 Da, in agreement with monoacetylation of H2B.A at a lysine residue between positions 1-25 (Figure 5C). The mass discrepancy could also indicate the presence of trimethylation (+42.05 Da, theor.); however, interrogation of several fragment ions revealed an average error of 13.9 ppm for trimethylation and 0.1 ppm for acetylation. Thus we conclude that peak 5 of Figure 1A consisted largely of monoacetylated H2B. A. Four c-type ions were also observed in the ECD spectrum that matched the H2B.F sequence (c26 shown in Figure 5C). This detailed study of the H2B gene products expressed to ∼3% relative abundance in HeLa cells identified seven distinct H2B variants and their modified forms (Table 1). In contrast to Tetrahymena, the HeLa cell H2B variants were not abundantly modified. There is no evidence for N-terminal methylation in human H2B. However, our work does confirm previous reports of acetylation of H2B.A with other monoacetylated variants likely lying below our detection limit. Compared to histones H3 and H4 in HeLa cells, H2B does not show a high level of modification complexity. The existence of different gene family members might represent an alternate mechanism by which the cells modulate chromatin function (e.g., in different tissues or at different stages of development), a possibility now under investigation. Most of the H2B forms observed here differ by only one or two amino acids. Some of the more interesting shifts include alanine (H2B.A) to serine (H2B.K/T) in the C-terminal domain and alanine (H2B.A) to a threonine (H2B.B) in the N-terminal domain. These serine and threonine residues can potentially serve as phosphorylation sites. Since the levels of phosphorylation of H1, H3, and H4 have been shown to be greatest during

Table 1. Different Forms of H2B Expressed in Asynchronous HeLa Cells gene family member

mass (Da) exp.b

mass (Da) theor.b

mass (Da) exp. w/labeled met b

PTM

H2B.J H2B.K/Ta H2B.A HAB.Q H2B.E H2B.B H2B.A H2B.F

13815.3 13814.8 13830.7 13844.6 13844.6 13860.4 13873.0 13873.5

13816.5 13814.5 13830.5 13844.5 13844.5 13860.5 13872.5 13874.5

13823.4 13823.6 13838.4 13853.3 13853.3 13868.4 13882.4 13882.8

0 0 0 0 0 0 monoacetyl 0

abundancec (from intact ion ratios)

9 ( 4%d 43 ( 5% 31 ( 5%d 11 ( 2% 6 ( 3%

abundancee (from fragment ion ratios)

3% 6% n.a. 18% 13% n.a. n.a. n.a.f

a These forms have the identical protein sequence. b All masses include intentional oxidation of two methionines to their respective sulfones (∆m ) +64 Da). c Abundances were calculated using protein ion relative ratios35 and are reported as percentages of the total H2B forms in the mixture. Precision values were determined from 4 measurements, with 2σ values reported. d The percent abundance is the sum of both H2B forms that are isobaric or within 2 Da of one another. e Abundances for these forms were calculated using a combination of protein ion relative ratios and fragment ion relative ratios. A manuscript validating this procedure using human histone H4 has been submitted.35 f Four fragment ions consistent with H2B.F were observed (c20, c25, c28, and c39).

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research articles mitosis, we interrogated M-phase and cell cycle stage-enriched samples. Marked dynamic changes in H2B modification during cell cycle progression do not appear to occur, since the H2B mass spectra for each cell cycle time point (mid-S, mid-G2, M/G2, and mid-G1) were quite similar to that of asynchronous HeLa cells (Figure 6). Phosphorylation, in particular, is not evident in these samples. Moreover, we found that samples from asynchronous and colchicine-treated cells samples have very similar ESI/Q-FTMS profiles (Figure 7B). Another cellular perturbation explored was overnight treatment with the histone deacetylase inhibitor sodium butyrate. This resulted in detectable increases in the abundance of mono-, di-, and triacetylated forms of H2B family members (Figure 7D). Upon MS/MS of H2B.A, +42.02 Da satellite peaks were confirmed to report on acetylation. Precise localization of monoacetylation was assigned to lysine 15 on H2B.A (Figure 7D). Ubiquitylation was not detected among any of the H2B variants analyzed above. This is likely due to ubiquitylated forms eluting apart from the major fraction of H2B in RPLC as employed here.

Conclusions Application of Top Down Mass Spectrometry and ECD have enabled better understanding of the nature of human histone H2B and the regulation of the expression and modification of H2B variants in HeLa cells. The high resolving power of FTMS in MS/MS mode combined with increased precursor ion populations afforded by the Quadrupole enhancement to FTMS makes Q-FTMS using ECD a desirable instrumental configuration to perform gene-specific protein identification in the Top Down approach.36 The measurements described here represent a significant advance for establishing the “basis set” of abundant forms of histone H2B isoforms expressed in human cells. The ability to dissect regulation of specific gene products by TDMS may prove more useful for H2B and H2A versus other human histones such as H3, where isoforms can be separated using chromatographic methods. Taken together, our combined work on human core histones has revealed that PTMs are far less abundant on bulk H2B and H2A than on H3 and H4, whereas the complexity of H2A and H2B due to coexpression of nonallelic variants is much greater than that of H3 and H4. High-resolution techniques such as Top Down Mass Spectrometry will prove useful in determining the significance of protein diversity created by gene families and/or PTMs in chromatin function and cell biology in general.36

Acknowledgment. The authors thank, Eric Thomas and Michael Boyne of the Kelleher group and Carol Nilsson and Christopher Hendrickson at the National High Magnetic Field FT-ICR MS Facility, Tallahassee, Florida (NHMFL, NSF, CHE94-13008) for technical assistance. James J. Pesavento was supported on an NIH Institutional NRSA in Molecular Biophysics (5T32 GM 08276). The laboratory of C. A. M. was supported by the University of Illinois and the Roy J. Carver Charitable Trust (grant #04-76) and the laboratory of N.L.K. by the Packard Foundation, the Sloan Foundation, and the National Institutes of Health (GM 067193-03). Supporting Information Available: Figure S1. IRMPD of H2B.A, H2B.J and H2B.K/T from the asynchronous HeLa cells. Figure S2. Sequence alignment of all the H2B variants present in asynchronous HeLa cells. This material is available free of charge via the Internet at http://pubs.acs.org. 238

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