Sensitive and Precise Characterization of Combinatorial Histone

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Sensitive and Precise Characterization of Combinatorial Histone Modifications by Selective Derivatization Coupled with RPLC-EThcD-MS/MS Rijing Liao, Dan Zheng, Aiying Nie, Shaolian Zhou, Haibing Deng, Yuan Gao, Pengyuan Yang, Yanyan Yu, Lin Tan, Wei Qi, Jiaxi Wu, En Li, and Wei Yi J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00788 • Publication Date (Web): 30 Dec 2016 Downloaded from http://pubs.acs.org on January 1, 2017

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Journal of Proteome Research

Sensitive and Precise Characterization of Combinatorial Histone Modifications by Selective Derivatization Coupled with RPLC-EThcD-MS/MS Rijing Liao,*, † Dan Zheng, ‡ Aiying Nie,⊥ Shaolian Zhou, ‡ Haibing Deng, ‡ Yuan Gao, ‡ Pengyuan Yang, § Yanyan Yu, ‡ Lin Tan, ‡ Wei Qi, ‡ Jiaxi Wu, † En Li, ‡ and Wei Yi*,‡ †

Xuhui Central Hospital, Shanghai Clinical Center, Chinese Academy of Sciences, Shanghai

200031, China ‡

Novartis Institutes for BioMedical Research (China) Co., Ltd., 4218 Jinke Road, Shanghai

201203, China ⊥

Thermo Fisher Scientific (China) Co.,Ltd., Building 6, No. 27 Xin Jinqiao Road, Shanghai

201206, China §

Department of Chemistry, Fudan University, 220 Handan Road, Shanghai 200433, China

KEYWORDS. combinatorial histone modifications, middle-down approach, LC-MS, derivatization, reversed-phase chromatography, EThcD

ACS Paragon Plus Environment

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ABSTRACT. Deciphering the combinatorial histone codes has been a long-standing interest in the epigenetics field, which requires the reliable and robust characterization of the posttranslational modifications (PTMs) coexisting on histones. To this end, weak cation exchangehydrophilic interaction liquid chromatography is commonly used in middle-down liquid chromatography-mass spectrometry (LC-MS) approaches for online separation of variously modified histone peptides. Here, we provide a novel strategy which combines the selective histone peptide derivatization using N-Hydroxysuccinimide propionate ester with reversed-phase (RP) LC for the robust, sensitive and reliable characterization of combinatorial histone PTMs. Derivatization amplifies the subtle physical differences between similarly modified histone peptides, thereby allowing base-line separation of these peptides by standard RPLC. Also, the sensitivity of MS is enhanced greatly by derivatization due to the increased peptide hydrophobicity and concentrated charge-state envelope during electrospray ionization (ESI). Further, we systematically optimized the dual electron transfer and higher-energy collision dissociation (EThcD) and achieved near-complete peptide sequence coverage in MS/MS spectra, allowing highly precise and reliable PTM identification. Using this method, we identified 311 and 293 combinations of histone H3 PTMs from the lymphoma cells Karpas-422 with/without drug treatment, confirming the advantages of our method in serving as a platform for profiling combinatorial histone PTMs.

INTRODUCTION Histones can be heavily modified with various post-translational modifications (PTMs) on their unstructured N-terminal tails.1 These PTMs can, as proposed by the histone code


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hypothesis, function in an independent or combinatorial fashion to dictate specific and distinct chromatin processes.1,2 So far, the functional implications of some individual PTMs on specific sites have been uncovered. For instance, histone H3 Lys-4 trimethylation (K4me3) has been associated with the transcriptional activation while histone H3 Lys-27 trimethylation (K27me3) has been associated with gene suppression.3 However, deciphering the combinatorial histone codes is hindered by the limited analytical technologies for the characterization of combinatorial PTMs. Middle-down mass spectrometry (MS) approaches, which analyze large peptides (typically larger than 3 kDa), is becoming a powerful tool to render the histone PTMs in a combinatorial fashion.4-9 In the case of histone H3 analysis, GluC is used to cleave H3 at the first glutamic acid in position 50 and generate a long N-terminal tail containing the 1-50 amino acids (1-50 a.a.). Since most histone H3 PTM sites are included in this tail, it is ideally suited to the investigation of combinatorial PTMs with reduced technical challenges from analyzing the intact histone H3 proteins. However, the complex PTMs on histone H3 still pose a formidable challenge to the analysis of this long tail. As multiple PTMs may occur on the same tail in various combinations, a sample from mammalian cells may contain thousands of long tails with the same sequence but different PTM patterns. These long tails are physically similar and therefore cause problems in their chromatographic separation. Without an effective separation, peptides with different PTMs will be introduced into MS simultaneously, leading to signal suppression of the low-abundance peptides. Moreover, the MS/MS spectra resulting from co-eluted isobaric peptides are almost impossible to be interpreted due to the lack of unique fragments.10,11 Thus, the chromatography method is the vital part of any middle-down approach and determines the sensitivity, efficiency and dynamic range of the overall analysis.6


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As one of the solutions, weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC), which is compatible with the highly hydrophilic peptides, has been successfully used in middle-down approaches for the separation of histone H3 tails. It resolves histone tails primarily by their distinct acetylation levels and secondarily by their different acetylation positions and methylation levels. Due to its high sensitivity to various acetylation states, it is particularly suited to those hyper-acetylated samples (e.g., histone samples treated with deacetylase inhibitor ). Using WCX-HILIC-based methods, hundreds of combinatorial PTM patterns on histone H3 had been reported by several pioneering studies.6-8 However, despite their success, these methods are now limited to several sophisticated laboratories, largely due to the special chromatography they use. Since the standard chromatography used in proteomics field remains the reversed-phase liquid chromatography (RPLC), many individual epigenetic laboratories are not ready to apply WCX-HILIC for routine use in view of the time, material and maintenance issues. Nevertheless, traditional RPLC only achieved marginal separation for long histone tails, prohibiting its use in the middle-down analysis for histones, especially for histone H3 with the most complex PTMs and being functionally critical in histone family. With RPLC alone, middle-down approaches identify only limited PTM species from the less complicated histones such as H2A, H2B and H4.9,12 We found that the poor RPLC separation could be dramatically improved by selective derivatization of the primary and secondary amines on histone tails with N-HydroxySuccinimide propionate ester (NHS-pro).13 The derivatized histone tails were more hydrophobic and can therefore interact much better with the stationary phase of RPLC. Furthermore, the subtle physical differences between histone tails were amplified greatly by this selective derivatization. We showed here that derivatization enabled RPLC to base-line separate various long histone H3


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tails with very similar PTM patterns. Additionally, the MS sensitivity also benefited from the derivatization. As a highly basic and long peptide, histone H3 tails display a wide charge-state envelope during electrospray ionization (ESI), resulting in a dilution of the overall signal. Derivatization can neutralize some positive charges on histone tail and concentrate the wide charge-state envelope. After derivatization, a dominant charge state representing the majority of the overall signal was yielded, allowing an increased detection sensitivity. Pinpointing PTMs from multiple possible residues on histone H3 tails requires almost a complete distribution of peptide backbone cleavage in MS/MS. Previous middle-down methods commonly used electron transfer/capture dissociation (ETD/ECD) for histone tail fragmentation. Although ETD/ECD can fragment peptide backbone more extensively than collision induced dissociation (CID) or higher-energy collision dissociation (HCD),14-16 it still failed to provide sufficient cleavages within the internal region of histone H3 tails. In this work, dual ETD and HCD (EThcD) was used in MS/MS to improve the fragmentation efficiency in the internal region. With optimized parameters, EThcD achieved near-complete peptide sequence coverage, allowing highly precise and reliable PTM site localization. The ETD reaction times used in EThcD MS/MS were dynamically adjusted based on the precursor intensities, expanding the dynamic range of the analysis. Based on the derivatization strategy and the improvement in MS/MS spectra, here we provided a novel RPLC-EThcD-MS/MS method for reliable, sensitive and precise characterization of the combinatorial histone H3 PTMs. Using a standard on-line nano-RPLC without the need for any salty solvent, our method can be readily applied and adapted in most proteomics laboratories. With this method, we identified 293 and 311 combinatorial histone H3 PTM forms from DMSO-


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or EI1-treated Karpas-422 cells (a human lymphoma cell line) with only 0.1 µg of histone tails, approximately one tenth of the amount used in previous WCX-HILIC methods.6-8 MATERIALS AND METHODS Chemicals and Reagents. Chemicals were purchased from Sigma unless stated elsewhere. The double deionized water was produced by Milli-Q gradient A10 system (Millipore, Bedford, MA). The standard peptides were purchased from GL Biochem Ltd (Shanghai, China). The NHS-pro reagent was synthesized as described previously.13 Mammalian Cell Culture and Histone Sample Preparation. Karpas-422 cells were obtained from the American Type Culture Collection and cultured in RPMI-1640 (Gibco, 11875) supplemented with 15 % (v/v) FBS (SAFC, 12003C), 100 U/mL Penicillin-Streptomycin (Gibco, 15140-122), in a 5 % (v/v) CO2 humidified incubator at 37 °C. Cells were treated with 5 µM EI1 or DMSO (as the control) for 3 days. Histone proteins were acid extracted as described by Shechter et al.17 The histones from acid extraction were further fractionated using a C8 column (150 mm × 4.6 mm, Agilent) with a gradient as follows: 0 % B for 5min, 0-35 % B from 5-10 min, 35 % B from 10-18 min, 35-65 % B from 18-70 min, (solvent A, 0.1 % TFA in H2O; solvent B, 0.1 % TFA in acetonitrile). Histone H3.2 purified was reconstituted in 100 mM NH4HCO3 (pH = 4) and digested with GluC (Roche Applied Science) at a protein: enzyme ratio of 7:1 for 4 hours at room temperature. The resulting peptides (1-50 a.a.) were purified by HPLC using a C8 column (250 mm × 4.6 mm, Agilent) with a flow rate of 1 ml/min and a gradient of 5-52 % B from 0-20 min (the same solvents used in histone fractionation). Fractions containing the histone tails (1-50 a.a.) were collected and dried in a SpeedVac. For propionyl-derivatization, histone tails were reconstituted in 50mM


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NH4HCO3 buffer (without any pH adjustment) at the concentration of ~0.1 µg/µL. Immediately before use, NHS-pro was dissolved in acetonitrile at the concentration of 200 mM. Equal volume of NHS-pro solution was added to the sample and mixed by the pipettor. The reaction was incubated at 50 °C for 30 min and thoroughly evaporated by a SpeedVac to remove any volatile remnants. Nano-RPLC-EThcD-MS/MS. For the optimization of EThcD, the derivatized histone tail (150 a.a.) was directly introduced into an Orbitrap Fusion (ThermoFisher Scientific) using a syringe at a flow of 300 nL/min. For cellular sample analysis, histone tail samples were loaded on a trap column (75 µm i.d., 2 cm, C18, 3 µm, 100Å, Dionex) and then separated with a RP column (75 µm i.d.,10.2 cm, C18, 3 µm, 120Å, New Objective) by an EASY-nLC 1000 (ThermoFisher Scientific). The LC gradient was as follows: 10-18% solvent B from 0 to 100 min at a flow rate of 300 nL/min (solvent A, 0.1 % formic acid in H2O; solvent B, 0.1 % formic acid in acetonitrile). The nano-ESI voltage was set to 2.1 kV and the capillary temperature was 275 °C. Each fullscan MS (m/z 700-760, resolution of 240 k) was followed with three EThcD-MS/MS (m/z 1502000, resolution of 240k) for the most intense three precursors. The maximum ion injection time (MIT) for MS1 and MS/MS were 50 and 1000 ms, and the automatic gain control (AGC) target for MS1 and MS/MS were 2 × 105 and 5 × 105 respectively. Two microscans per MS/MS. Only +8 precursor ions were isolated by the quadrupole with an isolation width of 1.2 Da. The dynamic exclusion time was set at 60 s. EThcD used 30% HCD normalized collision energy coupled with a varying ETD reaction time depending on the intensities of precursors (40 ms for the precursors < 1e6; 50 ms for those between 1e6 to 5e6; 60 ms for those between 5e6 to 1e7;


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70 ms for those > 1e7). The MIT of ETD reagent was set to 200 ms with ETD reagent AGC of 5 × 106. Data Analysis. Mascot Distiller (version 2.5.1.0) was used to extract, deconvolute and deisotope the MS and MS/MS spectra from the MS raw data. Then the processed data was searched by Mascot (Matrix Science, London, UK; version 2.5.0) against the human histone database exported from Uniprot. Enzyme specificity was set to GluC, allowing for up to 2 missed cleavages. Mass tolerance was set to 10 ppm for the precursor ions and 0.02 Da for fragment ions. Peptide isotopic error tolerance was set to 2 to eliminate peak detection error of C13 peaks. The propionylation (+56.0262 Da) on the N-terminus of peptides was set as a static modification. The variable modifications included acetylation (+42.0106 Da), propionylation (+56.0262 Da), monomethylation (+70.042 Da, the sum of propionylation and methylation), dimethylation (+28.0313 Da) and trimethylation (+42.0470 Da) on lysine; monomethylation (+14.0157Da) and dimethylation (+28.0313) on arginine; phosphorylation (+79.9663 Da) on serine and threonine. The peptide IDs with a Mascot ion score > 40 were accepted and further manually checked. In each replicate, the precursor intensity of every MS/MS spectrum with confident identification was extracted and summed. The abundance of each histone tail was expressed as its percentage in the total tails observed according to the following equation: percentage % = (MS1 intensity of the modified tail/ sum of all tails’ intensities) × 100 RESULTS AND DISCUSSION Derivatization Allowed High-resolution Separation With RPLC.


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Among the endogenous histone H3 PTMs, lysine methylations contribute the most to the diversity of PTM patterns, as it can commonly occur at multiple sites (K4, K9, K14, K18, K23, K27 and K36) with three states (mono-, di- or tri-methylation).18 However, histone tails that only differ in methylation levels or positions (hereinafter refer to as "methylation variants") are the most challenging species for LC to resolve. Unlike acetylation or phosphorylation which reduce the positive charges on histone tails, methylation has no impact on the charges of methylated residues. Thus methylation variants display not only the same number of charges but also the same intra-molecular charge distribution. Furthermore, compared with the histone tail, methyl-group is spatially minuscule and therefore conveys negligible changes in molecular sizes and shapes to methylated tails. Thus, these methylation variants are extremely similar in physical properties and therefore cannot be distinguished well by any chromatography.6 Our experiments showed that the histone H3.2 tails (1-50 a.a.) with acetylation or phosphorylation were able to be separated from other tails by RPLC, whereas the two methylation variants with K4 and K27 mono-methylation (K4meK27me) or K4 dimethylation (K4me2) failed to be resolved and coeluted ( Figure S1). Nevertheless, these physically similar methylation variants showed remarkable differences in the derivatization with NHS-pro. Chemically, the ε-amines of unmodified, mono-, di- or trimethylated lysines belong to primary, secondary, tertiary and quaternary amines respectively. Only primary and secondary amines can be propionylated by NHS-pro while the tertiary and quaternary amines remain unreacted. This phenomenon can be utilized to amplify the differences between methylation variants. For instance, during the derivatization of the three isomeric tails, the tail with K4meK27me added one more propionyl-group than the other two tails with K4me2 or K27me2 (Figure 1). As a result, it was more hydrophobic and thus could be distinguished


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easily by following chromatography (Figure 2). To the tails with K4me2 or K27me2, an identical number of propionyl-groups were added, however, the physical differences between them were still amplified due to the differential distributions of propionyl-groups. As propionylation can neutralize the positive charge of lysine, the distinct distributions of propionylation should lead to different intra-molecular charge distributions and therefore distinct molecular shapes or polarities.

Figure 1. An example of how derivatization amplifies the differences between methylation variants. Three isobaric methylation variants possess additional differeces in propionylation degrees or positions after derivatization.

To test the effect of propionyl-derivatization on RPLC separation, a mixture containing six synthetic histone H3.2 tails (1-50 a.a.) with K4me2, K27me2, K36me2, K4meK27me, K9 acetylation (K9ac) or S10 phosphorylation (S10pho) was derivatized with NHS-pro. Among them, 9 propionyl-groups were added to three tails (K4meK27me, K9ac and S10pho) while 8 propionyl-groups were added to the other tails (K4me2, K27me2 and K36me2), respectively. The derivatized mixture was then analyzed by a standard nano-RPLC-MS.


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Figure 2. RPLC separation of propionyl-derivatized histone H3 peptides (1-50 a.a.) with a 100-min gradient from 90% solvent A ( 0.1% fomic acid in H2O) to 20% solvent B ( 0.1 % formic acid in acetonitrile). The peptides with original K4 dimethylation, K27 dimethylation, K36 dimethylation, K4 and K27 monomethylation, K9 acetylation, or S10 phosphorylation were labeled as K4me2, K27me2, K36me2, K4meK27me, K9ac and S10pho, respectively.

As shown in Figure 2, all the six tails could be baseline separated, proving the advantage of derivatization in promoting the RPLC separation. As expected, the tails with 9 propionylations eluted later than those with 8 propionylations. Meanwhile, tails with the same number of propionylation were further separated from each other based on the PTM types and positions. Our derivatization strategy enabled RPLC to provide the sufficient separation of histone tails for a high-quality middle-down analysis. Derivatization Improved MS Sensitivity by Concentrating the Charge-state Envelope. Middle-down MS approach commonly suffers a reduced analytical sensitivity compared to the bottom-up approach. One of the reasons is that the long peptides will produce a wide chargestate envelope during ESI, resulting in a dilution of the overall peptide signal.19 Positive charges are localized primarily at the basic groups during positive-mode ESI.20,21 Histone H3 tail contains 17 basic groups (seven lysines, eight arginines, one histidine and one N-terminus) within its 50 residues, resulting in a high charge-density and, therefore, a strong charge-charge repulsion (Coulombic repulsion) between these basic groups. For the highly basic peptides such as histone tails, Coulombic repulsion plays a dominant role in determining their charge-state distributions.22 It makes histone tails take fewer charges than their number of basic groups.


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Meanwhile, it also expands the charge-state envelope of histone tails. As showed in Figure 3a, the native histone H3 tails displayed a broad charge-state distribution from +6 to +14 with the +10, +11, and +12 charge states being predominant.

Figure 3. Propionyl-derivatization concentrated the broad charge-state envelope of histone tails (1-50 aa) into a dominant charge state. (a) The charge-state distribution of native tails; (b) The charge-state distribution of derivatized histone tails.

As mentioned above, the positive charges on lysines and N-terminal amine can be neutralized by derivatization. As a result, the charge density and therefore the Coulombic repulsion would be reduced significantly. After derivatization, histone tails rendered a narrow envelope ranging from +6 to +9 with the +8 being the predominant charge state representing the majority of overall signal (Figure 3b). Apart from charge state manipulation, derivatization further facilitated MS sensitivity by increasing the hydrophobicity of histone tails, as the hydrophobic peptides generally ionize more efficiently than the hydrophilic ones.23,24 In combination with these advantages, as shown in our experiments, the signal intensity for derivatized histone tails was enhanced by 10-fold (Figure 3). EThcD-MS/MS Optimization. Histone H3 possesses more than a dozen of PTM sites spreading over its N-terminal tail. Ideally, it requires almost a complete distribution of peptide backbone cleavages in MS/MS to


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pinpoint PTM sites. Although ETD MS/MS can cleave peptide backbone in a relatively unbiased fashion, some preferential ETD cleavages have been observed by many studies.25,26 Our experiments showed that ETD preferentially cleaved the amino- and carboxyl-terminal regions of histone tails, generating the consecutive and intense fragment ions representing both termini. Conversely, it rarely cleaved backbone between 21th to 30th residues and produced very few fragment ions representing the internal region of tails (Figure 4a). The deficiency in c21-c30 and z21-z30 ions prevented the precise assignment of the modifications within this range. For instance, methylations on K23 or K27 were hard to be distinguished without the fragment ions deriving from the cleavages between K23 to K27.

Figure 4. MS/MS spectra of propionyl-derivatized histone H3 (1-50 aa) tail using (a) mere ETD with 50 ms reaction time or (b) EThcD with 50ms reaction time and 30% HCD energy.

More recently, EThcD combining both ETD and HCD has been used in the analysis of phosphopeptides and intact proteins to improve both efficiency and randomness of MS/MS fragmentation.27,28 The most crucial factors governing the EThcD spectra are HCD energy and ETD reaction time. We first fixed the ETD reaction time at 50 ms while varying the HCD energy from 0 to 50 %. The signal intensities of c232+, c272+, c282+, z222+, z262+, z312+ were investigated to assess the fragmentation efficiency in the internal region. As shown in figure 5a, EThcD


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enhanced the ion intensities in all cases. Especially, the c232+, c272+, c282+, and z312+ appeared only under EThcD mode, indicating the advantage of EThcD in providing more efficient cleavages in the internal region of tails. Since 30 % HCD energy could generally produce the most abundant and intense fragment ions, we then applied varying ETD reaction times (20-80 ms) coupled with a static HCD energy at 30 % in following experiments.

Figure 5. (a) Intensities of fragment ions generated by EThcD MS/MS using 50 ms ETD reaction time coupled with varying HCD energy (050%). (b) Total ion current (TIC) of 1+, 2+ or multiply charged fragment ions generated by EThcD MS/MS using varying ETD reaction times (2080 ms) coupled with 30 % HCD energy.

In the MS/MS spectra with short reaction times, a large amount of multiply charged fragment ions (MCFIs, z ≥ +3) which mainly consisted of the reduced charge precursors and the fragment ions containing >35 residues were observed. These MCFIs provided little information necessary for PTM site assignment while increasing spurious fragment ion matches. Furthermore, they complicated the spectra and often prevented the charge-state determination for nearby fragment ions. For instance, in a MS/MS spectrum using 20 ms reaction time, the charge state determination for z272+ (m/z 1514.35), a crucial fragment ion to distinguish the PTMs on K23 or K27, was prohibited by the wide isotopic envelope of z403+ (m/z 1511.19-1514.54), even though z272+ and its isotopic peaks had been detected clearly (Figure S2a). Conversely, the charge state


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of z272+ could be determined correctly in the MS/MS spectrum using 80 ms reaction time, as the isotopic envelope of z403+ had shrunk (m/z 1512.19-1513.54) (Figure S2b). Nevertheless, the signal intensity of z272+ was lower in the 80 ms spectrum than in the 20 ms one, suggesting that the longer ETD reaction time would also reduce the intensities of desirable ions. We then investigated the total ion current (TIC) for 1+, 2+ and MCFIs under varying ETD reaction times from 20 to 80 ms. Figure 5b shows that the TIC of MCFIs was deduced rapidly as the reaction time was increased while the TICs of 1+ and 2+ ions found their maximum at 40 and 30 ms, respectively. When the reaction time was increased beyond 40 ms, both TICs of 1+ and 2+ ions were deceased slightly. To provide MS/MS spectra of high quality, the ideal ETD reaction time should represent the best balance between eliminating harmful MCFIs and providing intense desirable ions. Given the wide concentration range of various histone tails in cells, the optimal ETD reaction time should not be fixed. For those precursors with low intensities (< 1e6), 40 ms reaction time should be the best as it yielded the maximal TIC for the useful ions while reducing the MCFIs modestly. Whereas those intense precursors that could tolerate slight reduction in fragment ion intensities, using the longer reaction times to remove more MCFIs may be optimal. Finally, a static 30% HCD energy coupled with the dynamic ETD reaction time depending on the precursor intensities enabled MS/MS to achieve near-complete peptide sequence coverage for histone tails over a wide concentration range (Figure 4b). Characterization of the Combinatorial PTMs on Histone H3 in Karpas-422 Cells. Enhancer of zeste homolog 2 (Ezh2) protein is the catalytic subunit of the polycomb repressive complex 2 (PRC2), which represses gene expression through methylation of histone H3 on lysine 27.29-31 PRC2 plays an important role in the epigenetic regulation and the aberrant expression or


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mutation of Ezh2 is associated with a broad spectrum of tumors.32-34 Karpas-422 cell line is a diffuse large B-cell lymphoma cell line possessing the Ezh2 Y641N mutation that leads to an abnormally enhanced methylation activity of Ezh2 on H3K27me2.35 In this study, we applied our middle-down method to investigate the combinatorial histone PTMs in Karpas-422 cells treated with DMSO or EI1,36 which is a potent and selective Ezh2 inhibitor ( Figure S3). To characterize and semi-quantitate combinatorial PTMs, 0.1 µg histone H3 N-terminal tails from Karpas-422 cells were derivatized and then analyzed by nano-RPLC-EThcD-MS/MS, in three biological replicates. As ions were dominantly +8 charged during ESI, the full-scan MS spectra were acquired within a narrow m/z range (700-760 Da) and the MS/MS spectra only targeted +8 charged precursors to avoid the repetitive acquisition of the same peptides with other charges. Because some isobaric peptides eluted in close proximity, it might be difficult to identify the boundaries of chromatographic peaks for these peptides. Thus, we used the MS1 intensity instead of extracted ion chromatogram (XIC) in peptide quantification. Every peptide intensity was normalized to the summed intensity of all the peptides identified in each biological replicate to eliminate the systematical errors such as uneven sample loading and uneven ESI efficiency between LC-MS runs. (see "Materials and Methods" for details). In total, 311 and 293 combinatorial PTM forms were identified from the EI1- or DMSOtreated Karpas-422 cells. The peptides that could be identified in all of the three replicates were shown in Table S1 and the peptides identified only in one or two replicates were presented in Table S2. Base on the data in Table S1, calculating the summed percentage of peptides with 0, 1, 2, 3, 4, or 5 PTMs indicated that the majority of peptides under both treatments contained 2 or 3 co-existing PTMs (Figure 6a). This preference for co-occurrence strongly supported that crosstalk mechanism between 2 or 3 modification sites may be prevalent for epigenetic regulation.


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Acetylations were observed at K9, K14, K18, K23 and K27, with K14 and K23 being the dominant sites ( Figure S4). Nevertheless, the overall acetylation degree of histone H3 in Karpas422 cells was not high. As shown in Figure 6b, about 60% peptides were not acetylated and ca. 30% peptides contained only one acetylation. In contrast, methylations were dominant PTMs in histone H3. Methylations were observed at K4, K9, K14, R17, K18, K23, R26, K27, K36, K37, R40 and R42, with K9, K27 and K36 being the most heavily methylated residues ( Figure S4). This result clearly demonstrated that our method is quite useful for histone middle-down analysis especially when methylation is the most prevalent cellular PTMs. Regardless of the abundant methylation on either K27 or K36, K27me3 was rarely found to co-occur with K36me3 in the same peptide, confirming the bidirectional antagonism between them that has been reported previously.7,37 When tracking the combinatorial partners for each single PTM, we also found K9 methylation, K23 acetylation or K14 acetylation frequently co-occurring with K27me3 in DMSO-treated sample (Table S1).


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Figure 6. The summed percentage of peptides (a) with n modified residues or (b) with n acetylations from either DMSO- or EI1treated karpas-422 cells. The error bars were established on the

basis of triply biological replicates.

In the DMSO-treated samples, a basal level of single K27me3 modification in Karpas-422 was observed as the most highly abundant PTM shown in the table S1. Totally about 38 % peptides in the DMSO-treated cells harbored K27me3 alone or in combination with other PTMs ( Figure S4 and Table S1). With EI1 compound treatment, the abundance of K27me3-containing peptides was reduced dramatically from 38 % to 13%, which was consistent with the function of EI1 as an Ezh2 inhibitor. Also K9me3 was observed as the second most abundant species in the EI-treated cells with almost 3-fold increase compared to DMSO treated samples (Table S1). And rare modifications such as methylations on R42 and phosphorylation on T45 appeared in the Table S2 indicating they are consistently low in their abundance. Intriguingly, single modification pattern on either R42 or T45 was not identified, suggesting these rare modifications may need to colocalize with other critical PTM markers for their epigenetic function. To our surprise, peptides


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bearing K27me3 with various PTM combinations displayed different sensitivities to the EI1 treatment (Table S3). Although the mechanism for the diverse EI1 sensitivity among combinatorial PTM forms remains unclear, the highly sensitive combinatorial PTM pattern, instead of global K27me3 level, may be potentially used as a more sensitive and specific biomarker to assess PRC2 inhibition. As Ezh2 inhibitors entered into clinical trials in recent years, our result would provide more mechanistic insights for details of drug action in cancer cells. CONCLUSION Selective propionyl-derivatization of histone H3 N-terminal tails dramatically improved the resolution of RPLC separation and sensitivity of MS analysis, thus, allowing a robust and sensitive RPLC-based middle-down analysis. The optimization of EThcD achieved the nearcomplete peptide sequence coverage in MS/MS, thereby improving the accuracy and confidence for PTM localization. Based upon these advantages, we identified 293 and 311combinatorial PTM forms from Karpas-422 cells treated with DMSO or EI1. We confirmed a dramatic decrease of K27me3 after EI1 treatment and the antagonism between K27me3 and K36me3. Additionally, we observed new candidate combinatorial partners of K27me3 and characterized diverse sensitivities of K27me3-containing peptides to PRC2 inhibition. Since our method used standard RPLC, it can be readily applied in most proteomic platforms to discover new combinatorial histone PTM partners and elucidate how histone modifications are dynamically established and erased. ASSOCIATED CONTENT


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Supporting Information. Figure S1: RPLC separation of standard histone H3.2 tails (1-50 a.a.) with a 100-min gradient from 100% solvent A ( 0.1% fomic acid in H2O) to 10% solvent B ( 0.1 % formic acid in acetonitrile). Figure S2: EThcD-MS/MS spectra of propionyl-derivatized histone H3.2 tails (1-50 a.a.) using (a) 20 ms ETD reaction time or (b) 80 ms ETD reaction time. Figure S3: The structure of EI1. Figure S4: The distribution of main PTMs on each residue of histone H3.2 in Karpas-422 cells treated with DMSO or EI1. Table S1: The combinatorial histone H3.2 PTMs identified from Karpas-422 cells treated with EI1 or DMSO in all of three biological replicates. Table S2: The combinatorial histone H3.2 PTMs identified from Karpas422 cells treated with EI1 or DMSO in only one or two replicates. Table S3: The peptides containing K27me3 identified in both EI1 or DMSO treated Karpas-422 cells. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *R. Liao: Phone: +86-21-54049262, Fax: +86-21-54049262, e-mail: [email protected]. *W. Yi: phone: +86-216-1606085, Fax: +86-216-1606626, e-mail: [email protected]. Present Addresses The current address of Shaolian Zhou is Grenzacherstrasse 124 4058 Basel Switzerland. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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This work was supported by the National Natural Science Foundation of China (21605154) and the MOST 973 project (2012CB910504). ABBREVIATIONS PTM, post-translational modification; LC-MS, liquid chromatography-mass spectrometry; RP, reversed-phase; ESI, electrospray ionization; EThcD, electron transfer and higher-energy collision dissociation; WCX-HILIC, weak cation exchange hydrophilic interaction liquid chromatography; NHS-pro, N-HydroxySuccinimide propionate ester; CID, collision induced dissociation; HCD, higher-energy collision dissociation; AGC, auto gain control; MCFIs, multiply charged fragment ions; TIC, total ion current; XIC, exacted ion chromatogram; Ezh2, Enhancer of zeste homolog 2; PRC2, polycomb repressive complex 2. REFERENCES (1) Strahl, B. D.; Allis, C. D. The language of covalent histone modifications. Nature 2000, 403, 41-45. (2) Jenuwein, T.; Allis, C. D. Translating the histone code. Science 2001, 293, 1074-1080. (3) Akkers, R. C.; van Heeringen, S. J.; Jacobi, U. G.; Janssen-Megens, E. M.; Françoijs, K. J.; Stunnenberg, H. G.; Veenstra, G. J. A hierarchy of H3K4me3 and H3K27me3 acquisition in spatial gene regulation in Xenopus embryos. Dev. Cell. 2009, 17, 425-434. (4) Taverna, S. D.; Ueberheide, B. M.; Liu, Y.; Tackett, A. J.; Diaz, R. L.; Shabanowitz, J.; Chait, B. T.; Hunt, D. F.; Allis, C. D. Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini. Proc. Natl. Acad. Sci. U S A, 2007, 104, 2086-2091.


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For TOC only:


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Figure 1. An example of how derivatization amplifies the differences between methylation variants. Three isobaric methylation variants possess additional differeces in propionylation degrees or positions after derivatization. 325x173mm (120 x 120 DPI)


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Figure 2. RPLC separation of propionyl-derivatized histone H3 peptides (1-50 a.a.) with a 100-min gradient from 90% solvent A ( 0.1% fomic acid in H2O) to 20% solvent B ( 0.1 % formic acid in acetonitrile). The peptides with original K4 dimethylation, K27 dimethylation, K36 dimethylation, K4 and K27 monomethylation, K9 acetylation, or S10 phosphorylation were labeled as K4me2, K27me2, K36me2, K4meK27me, K9ac and S10pho, respectively. 310x83mm (120 x 120 DPI)



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Figure 3. Propionyl-derivatization concentrated the broad charge-state envelope of histone tails (1-50 aa) into a dominant charge state. (a) The charge-state distribution of native tails; (b) The charge-state distribution of derivatized histone tails.


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Figure 4. MS/MS spectra of propionyl-derivatized histone H3 (1-50 aa) tail using (a) mere ETD with 50 ms reaction time or (b) EThcD with 50ms reaction time and 30% HCD energy. 293x187mm (120 x 120 DPI)



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Figure 5. (a) Intensities of fragment ions generated by EThcD MS/MS using 50 ms ETD reaction time coupled with varying HCD energy (0-50%). (b) Total ion current (TIC) of 1+, 2+ or multiply charged fragment ions generated by EThcD MS/MS using varying ETD reaction times (20-80 ms) coupled with 30 % HCD energy.


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Figure 6. The summed percentage of peptides (a) with n modified residues or (b) with n acetylations from either DMSO- or EI1-treated karpas-422 cells. The error bars were established on the basis of triply biological replicates. 173x186mm (120 x 120 DPI)


Sensitive and Precise Characterization of Combinatorial Histone

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