Quantification of Histone Modifications by Parallel-Reaction Monitoring

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Quantification of histone modifications by parallelreaction-monitoring (PRM) - a method validation James Sowers, Barsam Mirfattah, Pei Xu, Hui Tang, In Young Park, Chery Walker, Ping Wu, Fernanda Laezza, Lawrence C. Sowers, and Kangling Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02615 • Publication Date (Web): 10 Sep 2015 Downloaded from http://pubs.acs.org on September 14, 2015

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Analytical Chemistry

Quantification of histone modifications by parallel-reaction-monitoring (PRM) - a method validation James Sowers1, Barsam Mirfattah1, Pei Xu3, Hui Tang1, In Young Park2; Cheryl Walker2, Ping Wu3, Fernanda Laezza1, Lawrence C. Sowers1, Kangling Zhang1, *

1. Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, 77555 2. Institute of Biosciences & Technology, Texas A & M Health Sciences Center, Houston, TX 77030 3. Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas, 77555

To whom correspondence may be addressed: Kangling Zhang, Department of Pharmacology and Toxicology, UTMB at Galveston, TX 77554; Tel.: 409-772-9650; E-mail: [email protected]

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Abstract Abnormal epigenetic reprogramming is one of the major causes leading to irregular gene expression and regulatory pathway perturbations, in the cells, resulting in unhealthy cell development or diseases. Accurate measurements of these changes of epigenetic modifications, especially the complex histone modifications, are very important, and the methods for these measurements are not trivial. By following our previous introduction of PRM to targeting histone modifications (Tang, H.; Fang, H.; Yin, E.; Brasier, A. R.; Sowers, L. C.; Zhang, K., Multiplexed parallel reaction monitoring targeting histone modifications on the QExactive mass spectrometer. Analytical chemistry 2014, 86 (11), 5526-34), herein we validated this method by varying the protein/trypsin ratios via serial dilutions. Our data demonstrated that PRM with SILAC histones as the internal standards allowed reproducible measurements of histone H3/H4 acetylation and methylation in the samples whose histone contents differ at least one-order of magnitude. The method was further validated by histones isolated from histone H3 K36 trimethyltransferase SETD2 knock-out mouse embryonic fibroblasts (MEF) cells. Furthermore, histone acetylation and methylation in human neural stem cells (hNSC) treated with Ascorbic Acid Phosphate (AAP) were measured by this method, revealing that H3 K36 trimethylation was significantly down-regulated by six days of treatment with vitamin C.

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Introduction Though it has passed more than a decade since the appearance of mass spectrometry for the analysis of histone modifications,1,2 fast and accurate quantification of histone modifications in complex biological entities is still an analytical challenge.3,4 Previous quantification methods of histone modifications included the derivation method, the SILAC or 15N metabolic labeling method, the iTRAQ or TMT method, and the MRM/SRM method. Each method has its advantages and limitations. The derivatization method utilizes the amine acylation reaction to bring a small mass tag to the unmodified lysine residues whose chemical property is close to acetylation such as the frequently used propionyl anhydride or d6-acetylanhydride. The advantage of this method is its simple procedure for readout of histone absolute acetylation percentage, while the disadvantage is that its quantification accuracy largely depends on completeness of acylation reaction and uniform sample preparation among samples. Acylation with hydroxyl groups in serine or threonine and with monomethylated lysine further complicates the quantification. The SILAC or 15N method uses stable isotope labeling technique to incorporate heavy isotope labeled amino acids such as 13C615N4 Arg or all-15N into the proteins of one type of cells from the medium in which the heavy amino acids are added in to replace their normal counterparts that are for growing the other type of cells for relative comparison.5,6 Pairs of chemically identical peptides of different stable-isotope peptides can be differentiated in a mass spectrometer owing to their mass difference arising from isotopes. The ratio of peak intensities in the mass spectrum for such peptide pairs accurately reflects the abundance ratio for the two proteins. The same strategy can be applied for quantification of histone modifications.7-9 However, the disadvantage of this method is that the number of samples (or types of cells) for quantification is limited. Moreover, peptide isobaric isoforms cannot be differentially quantified 3



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because only the precursor ions are used for quantification. The iTRAQ or TMT method was also primarily developed for quantitative protein expression analysis utilizing the reporter ions generated from the isobaric mass tags that are attached to the N-terminal amines of peptides and ε-amines of lysines. The improvement of this method from SILAC or 15N metabolic method is its extended capability of comparing as many as 10 samples at once, for instance using TMT10.10 This method can also be used for quantification of histone modifications in some cases.11 However, the accuracy of this method is susceptible to the noise or co-eluted interferences.12 Because the quantification is only based on the relative ratio between reporter ions, peptide isobaric isoforms cannot be differentiated, disabling its potential for quantifying peptides with isobaric modifications. The MRM/SRM targeting method has adapted the long-standing mass spectrometry-based approaches for drug metabolism and pharmacokinetics (DMPK) studies to peptide analysis and protein quantification.13 It is the most sensitive mass spectrometry method for detecting ions due to significant reduction of background noise. This method is also suitable for quantification of histone modifications.11,14 Since MRM/SRM can only be realized on a triple quadruple mass spectrometer whose inherent low-resolution prevents it from differentiating HPLC co-eluted peptides whose precursor ion masses differ by less than two Dalton, many histone peptides with isobaric modifications can hardly be differentially quantified by this approach. A recently emerged parallel-reaction-monitoring (PRM) method that is employed on a high-resolution orbitrap instrument, such as the QExactive mass spectrometer, enhances significantly the selectivity of quantification by targeting product ions.15-18 In this case, isobaric peptides with undistinguishable precursor ions but distinguishable fragmentation ions can be differentiated and quantified. This is especially important for the quantification of histone H3 K27 methylation/acetylation and H3 K36 methylation which are coexisted in complex 4


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isobaric peptide mixtures. Based on the repertoire of HCD fragmentation spectra established by PRM on a QExactive mass spectrometer.19 In this report, we validated this method for quantification of histone modifications. With SILAC histones as the internal standards, PRM is a relatively reliable method for quantifying acetylation and methylation at most of the known sites of histone H3/H4 with satisfactory reproducibility.

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Experimental Procedures Culture of U87 cells. U87 cells purchased from American Type Cell Collection (ATCC) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (ATCC) and 1% antibiotic-antimycotic solution (Corning) in a 75-CM2 flask in a 37ºC, 5% CO2 incubator.

Culture of human neural stem cells (hNSCs) and Ascorbic Acid Phosphate Treatment. Most reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Human fetal NSCs (line K048, generously provided by Dr. C.N. Svendsen) were isolated from an 8-week fetal forebrain and expanded primarily according to what we described previously.20 Cells were cultured as free-ﬂoating “neurospheres” in 75-cm2 ﬂasks with growth medium containing DMEM (high glucose, L-glutamine)/Ham’s F12 (3:1; Invitrogen/Gibco, Grand Island, NY). This basic growth medium was supplemented with 15 mM HEPES, 1.5% D-glucose, penicillin/ streptomycin (67 IU/ml/67 µg/ml; Cellgro, Herndon, VA), 25µg/ml bovine insulin, 100 µg/ml human transferrin, 100 µM putrescine, 20 nM progesterone, 30 nM sodium selenite, 20 ng/ml recombinant human epidermal growth factor (EGF)(R&D Systems, Minneapolis, MN), 20 ng/ml recombinant human basic fibroblast growth factor (bFGF)(R&D Systems), 5 µg/ml heparin, 10 ng/ml recombinant human leukemia inhibitory factor (LIF)(Chemicon, Temecula, CA), and 2 mM L-glutamine. The human fetal NSCs were incubated with 8.5% CO2 at 37oC. Two thirds of the growth medium plus supplemental reagents were replaced every 3–4 days. The expanded neurospheres were dissociated into single cells once every 10 days with 0.025% trypsin and mechanical trituration. For the ascorbic acid phosphate treatment experiment, hNSCs were treated daily with 0 or 400 µM 2-phospho-L-ascorbic acid trisodium salt for the entire 10 6


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days of growth, or from the fourth day of growth to the tenth day. On the tenth day of each respective treatment, cells were collected, pelleted and frozen at -80oC until histone analysis was conducted.

Culture of MEF cells with SETD2 Knock-out. SETD2 flox/flox MEF parental MEF cells (B9, passage 29) and SETD2 flox/flox MEF transfected with ER-CRe, selected using Blastidin (4 µg/mL) (B4, mixed population) were grown in the medium (DMEM + 10% FBS + Glutamax). SETD2 knock-out was induced by 2 µM 4-hydroxy tamoxifen (TAM) from 10 mM TAM stock solution (Sigma, H7904-5mg in 1 mL ethanol) for five days. Cells treated with the vehicle were used as controls.

SILAC histone preparation SILAC MEM medium was prepared as following: 50 mg

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C615N4-Arginine·HCl (10R, 13C

Molecular, Inc. Fayetteville, NC) and 60 mg L-lysine (Sigma-Aldrich, St. Louis, MO) were dissolved in 2 mL H2O. After filtered through a 0.22 µM membrane, the amino acid solution was added to 500 mL SILAC medium (Thermo) with 5 mL antibiotic-antimycotic solution (Corning) and 50 mL SILAC FBS (Thermo). Cryopreserved U87 cells were seeded in 35-mm (diameter) culture dishes with SILAC MEM medium. After confluent, cells were split into one size bigger dishes. After several rounds of splitting, cells finally grew in 150-mm dishes till confluent. Cells were collected and heavy atom incorporation was measured by targeting histone proteins.

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Histone isolation and digestion. Histone purification was carried out using a standardized protocol as previously reported.19 Histone concentrations were estimated by Bicinchoninic Acid Protein Assay (BCA) (Pierce/Thermo-Fishers) and re-checked by comparing with the ESI signals of histone peptide standards. In all the tests performed in this manuscript, the usage of trypsin (Sigma) was constant, 1 µg, while histone proteins varied. Digestion time was 8 hours at 37 º C.

Histone peptide standards synthesis. All methylated peptide standards used in this study were synthesized by Peptide 2.0 Inc (Chantilly, VA). The peptides were purified by HPLC (purity > 99.5%) and their identities with modification information were validated by ESI-MS. The H3 and H4 peptides with sequences YRPGTVALR and VFLENVIR, respectively, were synthesized as previously described and used for normalization of histone concentrations and relative modification ratio calculation.

LC-Targeted MS-MS2 Experiments (PRM). The HPLC and mass spectrometry running conditions were the same as previously reported.19 The only difference here is that the inclusion list was doubly sized as equal amount of the precursor ions corresponding to the heavy peptides generated from SILAC histones were introduced as internal standards. Due to the slow scan rate of QExactive, the oversized inclusion list cannot be handled with one single mass spectrometry method even with multiplexed selection of retention time window, the inclusion list was divided into 2 to 3 sub-lists. The MS/MS spectra and the y-ions for quantification of modified peptides were referred to our previous publication.19 The only difference was that there was a ~10 Dalton mass increment for each arginine residue in SILAC peptides. 8


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Western blot analysis of histone modifications 10% SDS-PAGE gels were casted freshly in the laboratory using Bio-Rad MiniPROTEAN®Tetra Cell. Equal amounts of proteins (10 µg/lane) with DDT and loading buffer were heated at 95 °C for 5 minutes followed by electrophoresis using a Bio-Rad MiniPROTEAN® Electrophoresis system. After SDS-PAGE, proteins were transferred to a nitrocellulose membrane (Bio-Rad Trans-Blot TurboTM Blotting system). To block non-specific binding, the membranes were incubated in TBS with 0.1% Tween-20 (TBST) containing 5% BSA for 2 h. Subsequently, the membranes were incubated over night with antibodies against H3, K27 trimethylation, and K36 trimethylation, in TBST with 5% BSA. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG as secondary antibodies (1:2000 in TBST with 5% BSA) for 1 hour at room temperature. Detection was achieved using enhanced chemiluminescence (Luminata Classico Western HRP substrate). Quantification of band intensity was performed by AlphaImager software. Alternatively, histone extracts were lysed in extraction buffer containing 1:1 mixture of CelLytic™ M cell lysis Reagent (Sigma-Aldrich) and 2X Laemmli Sample Buffer (Bio-Rad, Hercules, CA) plus protease inhibitor cocktails, then resolved on SDS-PAGE and transferred to nitrocellulose membranes which were probed with specific first antibodies followed by appropriate fluorophore-conjugated secondary antibodies. The fluorescence signals were detected by LI-COR Odyssey imaging system (Lincoln, NE, USA). Band intensities were quantified by using Odyssey imaging software version 3.0.21

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Data processing Integration of peak area of each PRM pair defined for a specific modification was realized by Xcalibur Qual Browser. Relative histone modifications (the peak area of modified peptides are normalized to those of histone peptides that bear no modifications) and the modification fold changes or ratios were calculated using Microsoft Excel. Heatmap was using the R program as previously indicated.22

Results 1. Complexity of histone H3/H4 modified peptides can be resolved by product-ion scan or parallel-reaction-monitoring scan The success of quantification of histone modifications starts with good chromatography. Retention of the small and hydrophilic histone H3 lysine 4 methylated (H3 K4me) peptides on a mass spectrometry-favorable reverse-phase HPLC column is the barometer for good retention of many other histone modified peptides of interest with higher hydrophobicity. Typically, the core histone tryptic digests were separated by nano-HPLC with a 20 cm 360 (od) x75 (id) µm capillary column packed with 3 µm YMC-AQ particles with a linearly climbing gradient at 0.5% acetonitrile per minute starting from the starting point at 1% acetonitrile and flow rate 400 nL/min. Under this condition, H3 K4me peptides were eluted at ~15 minutes followed by other H3/H4 acetylation or methylation peptides and ended by the H3 K79me peptides (Figure 1). Based on this elution profile, a multiplexed PRM method was established with three major

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isolated retention time windows (RW1, 2, and 3). RW1 contains simply only H3K4me peptide, RW2 includes mainly H3K9, H3K27/K36, H4K20 methylated peptides, and RW3 includes H3/H4 acetylated peptides, H3K79 methylated peptides, and H3/H4 peptides without known modifications that were chosen for normalization of histone contents. RW2 that contained the majority of H3/H4 modified peptides chosen for quantification could be additionally isolated into multiple smaller isolated retention windows to enhance selectivity when the reproducibility of chromatography was guaranteed between runs. However, even under such nearly optimum condition, H3K27/K36 methylated peptides comprising of a mixture of nearly 30 peptides with un-, mono-, di-, and tri-methylation randomly distributed on K27 and K36 sites, could not be fully separated. Many of these peptides are isobaric isomers as labeled by a series of “*” in Figure 1A. Their corresponding triple-charged precursor ions are listed in the table below the figure followed by their corresponding peptide sequences with clear annotation of methylation configurations as shown in Figure 1B. All of these peptides were co-eluted within 1 minute and could not be differentiated by chromatographic characteristics and full-mass scan mass spectrometry. Production-ion scan or parallel-ion-scan is an irreplaceable resource to unambiguously differentiate these peptides.19 Trypsin digests not only un-methylated lysines, but also mono-methylated lysines, albeit with decreased degree. The digestion efficacy depends on protein/enzyme ratio and the incubation time of digestion. As shown by underlined sequences in Figure 1B, two thirds of H3K27/K36 methylated peptides are un- or mono-methylated at either K27 or K36. Inconsistency of digestion efficiency of un- and mono-methylated lysines, resulting from unequal concentration of proteins amongst samples that are digested with a fixed amount of trypsin to cause variable protein/enzyme ratio, is the major source of analytical variation which presents a challenge for accurate quantification of these peptides. 11



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2.Quantification dynamic range and lowest-of-quantification (LOQs) An important advantage of the PRM targeting methods over other methods is its high throughput and high accuracy. The quantification is based upon the product ions on a high resolution mass spectrometer. In order to evaluate the quantification limit and linear dynamic range, we synthesized a series of representative histone H3/H4 methylated peptides, most of which are dimethylated. As indicated in Figure S1, linear signal-concentration responses were obtained within 3 to 4 orders of magnitude and LOQs were in the range of 10 to 100 femtomole on the column. To note, the signal-to-concentration responses of H3K27/K36 peptides were about oneorder magnitude lower, which is approximately equivalent to one-order magnitude of higher LOQ, than other peptides. The cause for this difference may be due to the major factor: H3K27/K36 peptides in the form of triply-charged precursor ions and doubly-charged product ions have poorer electrospray signals than other peptides that generally produce doubly-charged precursor ions and singly-charged product ions.

3. Quantification of histone peptides using SILAC histones as internal standards The major concern of quantification accuracy is whether histone protein in each sample, within a group of samples to be relatively compared, proportionally produces a peptide which is chosen for quantification after trypsin digestion that linearly corresponds to its precursor-protein concentration. Deviation of quantification accuracy comes from the inconsistency of protein digestion efficacy among samples when peptides are targeted for implementing the 12


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quantification. As protein/protease ratio at a given digestion time is the major factor to govern protein digestion efficacy, the deviation of quantification accuracy can hardly be corrected by peptide internal standards. To cope with this problem, a protein internal standard (IS), such as SILAC histones, shows the promise of compensating this deviation when IS and samples are digested and analyzed simultaneously. To this end, we grew U87 cells in 6C134N15-arginine MDEM medium for at least 5 times of passing to ensure nearly 98% incorporation of heavy arginine into proteins (Figure S2). After their concentrations measured and heavy/light ratio determined to meet the requirement, the histone extracts were adequately aliquoted and saved for using as internal standards which were also termed here as 10R-histones. We prepared a series of nine U87 histone samples whose concentrations ranged from the lowest 0.13 µM, defined as sample #1, to the highest 4.16 µM defined as sample #9, forming a series of dilution with the ratio 1:2:4:8:12:16:20:24:32. 20 µl of 10R-histones whose protein concentration was about 0.19 µM was added to each sample vial. After adjustment of pH to ~8 by 0.1% NH3·H2O, histones were digested with 1 µg of trypsin at 37 °C for 8 hours. The digests, after vacuum dried, were dissolved in 50 µl of 1% formic acid before they were ready for LC-MS/MS analysis. The Orbitrap instrument was setup to do MS/MS against the inclusion list of precursor ions representative of typical histone H3/H4 acetylated and methylated peptides as previously reported 19. As indicated in Figure 2A, the H3 peptide YRPGTVALR signal intensities responded non-linearly to H3 concentration with severe ion suppression at high concentrations. However, with a correction by internal standard peptide Y10RPGTVAL10R (IS), a linear responsiveness between measured intensities and prepared concentration was established. Many modified peptides including H3 K18/K23 acetylation, H3 K4 and K9 methylation, H4K20 methylation had the same performance as the H3 peptide and, therefore, the ratio of modified 13



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peptides over H3 peptide could keep constant (fold change = 1 ± 0.2) without IS. Because these peptides behaved with the same features as the H3 peptide, after normalization, variations from both the numerator and the denominator were cancelled out. This “perfect” situation without internal standards cannot be applied to other modification sites, especially the H3 K27/K36 modification complex. For example, the signal intensities of H3K27me2K36me2, H3K27me3K36me2, H3K27me2K36me3, and H3K27acK36me3 peptides, were severely suppressed as protein concentrations increased (Figure 2C and D). However, such dissonance of signal responsiveness to peptide concentration were corrected by the IS peptides (Figure 2C &D). As a result, 10R-histones used as internal standards (IS) significantly improved quantitation accuracy as shown by Figure 3A & B from openly-dispersed spots on the graph and 38% overall mean RSD (relative standard deviation) without IS to narrowly-confined in the region of fold changes between 0.8 -1.2 and 18% overall mean RSD with IS, based on the quantification data at nine concentration of 43 PRM ion-pairs as shown in Table S1. It would be worthy to note that using single product ion for quantification did not yield sufficiently higher deviation from accuracy than using multiple product ions. To this end, the y112+ ions of H3 K27/K36 peptides, which have the highest intensity and no interference from other product ions with overlapped mass values, were solely chosen for quantification of H3 K27 and K36 modifications. Peptides with un- or mono-methylated lysines could be partially digested by trypsin, resulting in two peptides to be detected by mass spectrometry, one peptide with uncleaved lysine or monomethylated lysine and the other one with large amino sequence while the small amino sequence escaped the detection by mass spectrometry with the mass scan range set from 350 to 1500 for precursor ions. As shown in Figure 2D, the signal of the H3K27me3K36me peptide with the sequence,

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Kme3SAPATGGV36Kme1KPHR, ascended non-linearly with increasing protein 14


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concentrations, while the signal of the trypsin-cleaved peptide with the sequence, 27

Kme3SAPATGGV36Kme1, descended with increasing protein concentrations, indicating that

trypsin cut monomethylated H3 K36 (H3 K36me1) more efficiently in samples containing low concentration histone H3 than in samples containing high concentration histone H3. Without considering the differential ESI signal responses between these two peptides, nearly 100% of H3 K36me1was cut by trypsin at low concentration while ~20% at high concentration. To have an insight into the difference of ESI efficiency between these two peptides, we synthesized these two peptides and prepared their standard solutions with equal concentrations. They were analyzed together with other standard peptides as Figure S1 showed. The difference was gorgeous. The signal ratio between the peptides, H3 27Kme3SAPATGGV36Kme1 versus 27

Kme3SAPATGGV36Kme1KPHR, was more than 200 at their low concentrations (0.1nmol/mL).

This ratio sequentially decreased along with the increase of peptide concentrations and dropped down as low as 30 at the concentrations of 100 nmol/mL (Figure 3C). Taking into account of such differential ESI responses between these two peptides, the real % digestion at H3 K36me1 would be significantly lower than shown by the curve in Figure 3D. Since the added H3 K36me1 IS concentration could not always be the same as the H3 K36me1 sample there would because significant inconsistency of digestion efficacy at monomethylated H3 K36 between IS and the sample. The quantification of H3 K36 monomethylation would not be as accurate even with an internal standard peptide. In order to prevent monomethylated lysines from digestion by trypsin, we incubated a second set of samples prepared exactly the same as the one described above with deuterated acetyl-anhydride to add 2H3-acetyl groups to both un- and monomethyated lysines. However, the extraordinary complexity of peptide mixtures with both natural and chemical modifications made the quantification almost impossible (data not shown). It seemed 15



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that derivatization might not be an ideal procedure for the PRM method described here for quantification of histone H3 K27/K36 modifications. A plausible solution to this challenge would be to ensure approximately equal concentrations of samples to be analyzed so as to eliminate variation of digestion efficacy.

4. Verification of the PRM method with histone samples of known modification status Further, we test this method by analyzing histone modifications in MEF cells in which histone H3 K36 tri-methylation specific methyltransferase SETD2 was knocked out by SETD2flox/flox ERCre. The knock-out (KO) expression of SETD2 was induced by 4-hydroxy tamoxifen. Both SETD2 f/f (-/-) cells and SETD2 f/f (+/+) cells grown in the MDEM medium with either vehicle (alcohol) or 2 µM 4-hydroxy tamoxifen (TAM) were used for this study for comparing histone modification patterns by PRM. Calculation of histone modifications were based on the relative ratio of the intensity of a modified peptide to that of a H3 or H4 peptide that does not bear any modifications with or without the consideration of SILAC histones as internal standards. As demonstrated in the heat map (Figure 4A) and its originally associated data (Table S2 and Figure S3-S5), histone H3 (including both H3.1/H3.2 and H3.3 forms) K36 tri-methylation was significantly reduced in TAM-treated SETD2 f/f (+/+) cells as compared with vehicle-treated cells. However, H3 K36 trimethylation did not change in SETD2 f/f (-/-) cells treated with either vehicle or TAM. The two calculations with or without consideration of IS gave the same results. However, IS significantly improved the quantification accuracy as the mean RSD decreased from larger than 15% in the calculation without IS to ~10% with IS. The reduction of H3 K36 trimethylation in SETD2 KO MEF cells was validated by Western-blot analysis using an 16


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antibody against H3 K36 tri-methylation (Figure 4B, Figure S6). Under the same condition, H3 K27 tri-methylation did not decrease, which was consistent with the mass spectrometry measurements (Figure 4B, Figure S6).

5. Quantification of histone modifications in human neural stem cells (hNSCs) treated with vitamin C-Vc-or Ascorbic Acid Phosphate (AAP) 20 µl of core histones isolated from NSC with concentration of 1 mg/mL determined by BCA were mixed with 5 µl of SILAC histones before adding 1 µg of trypsin for 8-hour digestion. The digests were analyzed by PRM as above described. A total of 37 modified peptides were successfully quantified from the pre-set inclusion list that contains the precursor ions of both the targeting modified peptides and their heavy (10R) peptide counterparts. The percentage of each modification was calculated by the division of integrated peak area of individually targeted modified peptide by the integrated peak area of a peptide unique to either H3 or H4 peptide. When internal standards were applied in calculation, the peak area of each targeted peptide was normalized against the peak area of its 10R-labeled peptide that was produced by the digestion of the SILAC histones. Based upon the percentages of modifications, the ratios of Vc-treated over Vc-untreated (controls) were obtained. As demonstrated in Figure S7 & S8, the majority of modifications did not change with AAP-treatment for either the full ten days or the last six days. However, histone H3.1/3.2 K36 tri-methylation, histone H3.3 di- and tri-methylation, and H3.3 K27 tri-methylation were significantly reduced in the 6-day AAP treated hNSC, and only histone H3.1/3.2 K36 tri-methylation was slightly reduced in the 10-day AAP-treated hNSCs. Again, IS significantly improved the quantification accuracy as the mean relative standard deviation (RSD 17



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%) decreased from ~ 25% in the calculation without IS to less than 15% with IS (Table S3). A heat map as shown in Figure 5A was graphed based on the content of table S3 clearly illustrated the changes of histone modification patterns of hNSCs with vitamin C treatments. The bottom green zone revealed the reduction in H3 K27/K36 methylation of hNSCs treated with vitamin C. The significant reduction of H3 K27/K36 trimethylation in the 6-day AAP-treated hNSCs was validated by Western blot analyses carried out independently in two laboratories using antibodies against H3 K27 trimethylation and K36 trimethylation (Figure 5B & C).

Conclusion We have tested the solidity of the PRM method for quantification of histone modification by a dilution experiment with varying protein/trypsin ratio. We found that ESI signals of histone peptides did not linearly respond to peptide concentrations in the testing range of H3 protein from 0.13 µM to 4.16 µM that was digested by a fixed amount of trypsin (1µg). This nonlinearity, which might be mainly caused by protein concentration depended digestion efficiency and signal suppression, could be, at least partially, corrected by internal standards of the heavy peptides digested from SILAC histones. Since histone modifications were measured by the ratio of the integrated peak area over the integrated peak area of histone peptides that did not bear modifications, the inconsistency of measurements from samples to samples was significantly minimized to enhance reproducibility. As comparing the dilution experiments with the test/application experiments (SETD2 KO and hNSCs), we found the accuracy of the latter (Table S2 and S3) was better than the former (Table S1). An explanation could be that the amount of SILAC histones IS was close to the lowest concentration in the 9 dilution samples and IS might 18


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not be able to fully correct variation for the high concentration samples, while in the application samples, the amount of IS samples was consistently added to individual test samples whose concentration were close to each another. In order to achieve best accuracy, the sample concentration of samples and the SILAC histone concentrations added to the samples should be close so that discrepancy of trypsin digestion efficiency among samples and internal standards can be maximally reduced.

Without derivatization of unmodified lysines and using trypsin as

the protease to digest histone proteins, quantitative information on unmodified lysine states was lost. However, using Arg-C as the protease to digest histone proteins, both unmodified and modified lysine states could be quantified. In this case, the quantitative sensitivity for modified lysines would be decreased as trypsin is always the most highly efficient protease. In contrast, in order to achieve complete digestion of unmodified lysines to lessen its interference with quantification of modified lysines, a combination of trypsin and Lys-C would be considered.

The reliability of using PRM method for quantification of histone modifications was attested by its accurate measurement of H3 K36 tri-methylation that was depleted in SETD2 KO MEF cells. Further, this method was successfully applied to analyzing histone modification pattern rearrangement in hNSCs after they were treated with AAP. We observed a significant reduction of histone H3.1/3.2 K36 tri-methylation, H3.3 K36 di- and tri-methylation, and H3.3 K27 trimethylation in the final six days of AAP-treated hNSCs but not in the full 10 days of AAP treatment. AAP has been previously demonstrated to cause significant reduction of histone H3 K36 di- and tri-methylation in MSC through activation of ascorbate dependent histone demethylase KDMT2 and suggested that it would play a role in enhancing proliferation of stem cells.23,24 In our case as demonstrated here, H3 K36 di- and tri-methylation was also significantly 19



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Page 20 of 30

reduced in six days of AAP-treated hNSCs. Whether AAP induced histone H3 K36 demethylation in hNSCs was through the same mechanism as KDMT2 activation in MSC,23,24 or with additional involvement of SETD2 inactivation is currently not known. Since the antioxidant vitamin C stabilizes Fe2+, an essential cofactor for many demethylases, demethylation of histones would be expected for all lysines in vitamin treated cells. However, in our experiments and others’, vitamin C only significantly reduces H3 K36 tri-methylation, implying that either vitamin C does not have an equal effect on every demethylase or the expression of H3 K36me3 demethylase KDMT2 in stem cells is highly regulated by vitamin C.

To address these

unanswered questions, further studies on enzymatic assays followed by functionality investigation are needed. In summary, through the dilution experiments, the test and the application experiments, we have obtained instructive information regarding how the accuracy of PRM measurements can be assured and what precaution may be needed for sample preparation in order to obtain reliable quantification results of histone modifications in multiple samples assayed simultaneously.

ASSOCIATED CONTENT Supporting Information. Figures S1- S8, Table S1 & S2. AUTHOR INFORMATION Corresponding Author

20


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*To whom correspondence may be addressed: Department of Pharmacology and Toxicology, School

of Medicine,

UTMB,

Galveston,

TX 77554;

Tel.:

409-772-9650;

E-mail:

[email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported in part by the National Institutes of Health, National Cancer Institute (CA184097) and in part by the start-up fund to K. Z. provided by Department of Pharmacology and

Toxicology,

School

of

Medicine,

University

of

Notes The authors declare no competing financial interests.

21


Texas

Medical

Branch.


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Figure Legends Figure 1. Separation of core histone trypsin digest by nano UPLC. A. The elution profile of modifed peptides of interest and H3 /H4 peptides used for nrmalization for relative quantification. B. Listed all the possible methylated H3K27/K36 peptides eluted at ~30 minutes labeled as a serires of "*" with corresponing presursor ions as showed in the table below A. Underlined sequences with un- or mono-methylated lysines can be digested by trypsin.

Figure 2. ESI signal response of histone peptides to protein concentration with fixed amount of trypsin. A. ESI signal responses of the y 5 and y6 ions fragmented from H3 peptide with the sequence YRPGTVALR to the protein concentration. The responses were corrected by an internal standard with the same sequence generated from SILAC hsitones. B. ESI signal responses of the y 112+ ions fragmented from H3 K27/K36 peptides di- and tri-methylated, repectively at either site, to the protein concentrations. C. ESI signal responses of the y 112+ ions fragmented from H3K27/K36 peptide acetylated K27 and di-methylated at K36, repectively, to the protein concentrations. D. ESI signal responses of the y 112+ ions fragmented from H3K27/K36 peptides di-methylated at both K26 and K36, its isobaric peptide trimethylated at K27 and monomethylated at K36 and its trypsin-cut at K36 peptide to the protein concentrations. Each datapoint was an average of three technical repeats with standard deviation as shown by error bars on the curves.

Figure 3. Quantification accuracy

22


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Page 23 of 30

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A & B. Quantification of the modifications of interest as shown in Table S1 without IS & with IS. C. Differential ESI signal response between the long peptide H3K27me3K36me1 (trypsin un-cut) and the short peptide H3K27me3K36me1 (trypsin cut). D. Percent of trypsin cut H3K27me3K36me1peptide. Each datapoint is an average of three technical repeats. No error bars were shown for simplicity.

Figure 4. Histone modifications of MEF cells A. A heatmap of histone modifications of MEF cells. SETD2 f/f (+/+) - TAM: MEF cells transfected with SETD2 fox/fox Cre/ER construct without induction by TAM (SETD2 +, Control1); SETD2 f/f (+/+) + TAM: MEF cells transfected with fox/fox Cre/ER construct. Knock-out of SETD2 is induced by TAM (SETD2 -); SETD2 f/f (-/-): MEF cells transfected with empty fox/fox Cre/ER construct (SETD2 +, Control2); SETD2 f/f (-/-) + TAM: MEF cells transfected with empty fox/fox Cre/ER construct and treated with TAM (SETD2 +, Control3). B. Western-blot analysis of H3 K27/K36 tri-methylation.

Figure 5. Histone modifications of hNSC cells A. A heat map of histone modifications of NSC cells. NSC: neural stem cells grew without vitamin C for 10 days (Ctl: Control); NSC D4-10: NSC was treated with 400 µM vitamin C during day 4th to 10th; NSC D1-10: NSC was treated with 400 µM vitamin C during the whole 10 day period. Peptide numbers are referred to Table S3. B. A representative Western blot analysis (WB) of H3 K27 tri-methylation and H3 K36-trimethylation. C. Average of fold changes of H3 K27/K36 tri-methylation of three WB. Data presented are the averages of 3 independent experiments (mean ± sem) (* p < 0.05, ** p < 0.01, Student’s t-test). 23



Figure 1

VFLENVIR

YRPGTVALR

A

[M+2H]2+: 495.2926

H3

100 80

H3K79me

60

H4K16Ac

Relative Abundance

H4

[M+2H]2+: 516.8011

H3K18/K23Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 30

40 20 0

H3K4me 14

16

18

20

22

24

26

28

30 32 34 36 Time (min)

38

RW2

RW1

40

29.48 29.59 30.12 30.27 30.27

B H3.1/H3.2 27K

me0SAPATGGV

27K

me1SAPATGGV

44

46

48 50

RW3 RT(min)

27K 36 * me1SAPATGGV Kme1KPHR ** 27Kme2SAPATGGV36Kme1KPHR *** 27Kme3SAPATGGV36Kme1KPHR **** 27Kme3SAPATGGV36Kme2KPHR ***** 27Kme3SAPATGGV36Kme3KPHR

42

[M+3H]3+ H3.1/3.2, 487.9595, 492.6315, 497.3037, 501.9756, 506.6473 ,

H3.3 493.2914 497.9633 502.6352 507.3071 511.9790

* ** *** **** *****

36K 27K 36 me2KPHR me2SAPATGGV Kme0KPHR 36K 27K 36 27 36 me2KPHR me3SAPATGGV Kme0KPHR, Kme0SAPATGGV Kme3KPHR 27K 36 27K 36K SAPATGGV KPHR me2SAPATGGV Kme2KPHR me1 me3 27K 36 me2SAPATGGV Kme3KPHR

H3.3 27K 36 * me1SAPSTGGV Kme1KPHR ** 27Kme2SAPSTGGV36Kme1KPHR *** 27Kme3SAPSTGGV36Kme1KPHR **** 27Kme3SAPSTGGV36Kme2KPHR ***** 27Kme3SAPSTGGV36Kme3KPHR

27K

me0SAPSTGGV

27K

me1SAPSTGGV

36K 27K 36 me2KPHR me2SAPSTGGV Kme0KPHR 36K 27K 36 27 36 me2KPHR me3SAPSTGGV Kme0KPHR, Kme0SAPSTGGV Kme3KPHR 27K 36K 27K 36K SAPSTGGV KPHR SAPSTGGV KPHR me2 me2 me1 me3 27K 36 me2SAPSTGGV Kme3KPHR

24


Page 25 of 30

Figure 2

40

C

H3_y5 (IS) H3_y6 (IS)

Signal (Fold Change)

H3_y5 (No IS) H3_y6 (No IS)

30

40

H3K27acK36me2 (IS)

y = 0.8947x + 0.7653 R² = 0.9862

H3K27acK36me2 (No IS)

y = 0.9023x + 0.8414 R² = 0.9836


A

y = 1.0753x0.8355 R² = 0.9936

20

y = 1.0888x0.8267 R² = 0.9935

10

30

y = 0.8262x + 0.3533 R² = 0.9836

20

10 y = 0.945x0.5221 R² = 0.9689 0

0 0

10

20

30

0

40

B

40

20

30

40

D

H3K27me3K36me2_y11 (2+) (IS)

140

H3K27me2K36me2 (IS)

H3K27me2K36me3_y11 (2+) (IS)

H3K27me3K36me1 (IS)

120

H3K27me3K36me2_y11 (2+) (No IS)

H3K27me2K36me2 (No IS)

H3K27me2K36me3_y11 (2+) (No IS)

H3K27me3K36me1 (No IS)


30

10

Protein Conc. Ratio (Prepared)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


y = 0.7103x + 0.5637 R² = 0.981

20

y = 0.552x + 0.2682 R² = 0.9801 y = 1.0701x0.6532 R² = 0.9324

10

y = 1.103x0.458 R² = 0.8739

100

H3K27me3K36me0/1Cut

80 60 40 20 0

0 0

10

20

30

0

40 -20


10

20

30

Protein Concentration (Fold Change)

25


40


Figure 3

A

- IS

2

C

350

Diferential ESI signal response H3 27Kme3SAPATGGV36Kme1

300

[486.2978 (2+)

1.6

ESI Signal Ratio

Normalized Signal Ratio

1.8

1.4 1.2 1 0.8 0.6 0.4

643.3774 (y7)]

over H3 27Kme3SAPATGGV36Kme1KPHR

250

[(497.3036(3+)

200

581.3462 (y112+) ]

150 100 50

0.2

0

0

0 0

10

20

30

40

50

40

80

120

Concentration (nmol/mL)

Protein Conc. Fold Changes

B

100

D

+ IS

2

K27me0/1 Trypsin Cut K36me1 Trypsin Cut

80

1.8 1.6

% Digestion

Normalized Signal Ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 30

1.4 1.2 1 0.8

60

40

0.6 20

0.4 0.2

0

0 0

10

20

30

40

0

50

10

20

30

Protein Concentration (Fold Change)

Protein Conc. Fold Changes

26


40

Page 27 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4

A

H4 20Kme2VLR H4 20Kme1VLR H3 EIAQDF79Kme1TDLR H4 20Kme3VLR H3 EIAQDF79Kme2TDLR H3.1/3.2 27Kme1SAPATGGV36Kme2KPHR H3.1/3.2 27Kme2SAPATGGV36Kme2KPHR H3.1/3.2 27Kme3SAPATGGV36Kme2KPHR H3 9Kme2STGG14KacAPR H3 18KQLAT23KacAAR H3 9Kme1STGG14KacAPR H3.1/3.2 27Kme2SAPATGGV36Kme1KPHR H3 9KacSTGG14KacAPR H4 GLG12KacGGA16KacR H3 18KacQLAT23KacAAR H4 GG8KacGLG12KacGGA16KacR H3 T4Kme2QTAR H4 GGA16KacR H3.1/3.2 27Kme3SAPATGGV36Kme1KPHR H3 9Kme3STGG14KacAPR H3 T4Kme1QTAR H3.1/3.2 SAPATGGV36Kme3KPHR H3.1/3.2 27Kme2SAPATGGV36Kme3KPHR H3.1/3.2 27Kme1SAPATGGV36Kme3KPHR H3.1/3.2 27Kme3SAPATGGV36Kme3KPHR H3.3 27Kme3SAPSTGGV36Kme2KPHR H3.3 SAPSTGGV36Kme3KPHR

B

SETD2 f/f (-/-)

SETD2 f/f (+/+) - TAM

+ TAM

- TAM

+ TAM

K36me3 K27me3

H3

27



Figure 5

A H3 EIAQDF79Kme1TDLR H3 EIAQDF79Kme2TDLR H3 T4Kme1QTAR H4 20Kme2VLR H3 18KacQLAT23KacAAR H3.1/3.2

27K 36 me2SAPATGGV Kme2KPHR

H3.1/3.2


H3.1/3.2


H3.1/3.2 27KacSAPATGGV36Kme2KPHR H3 18KQLAT23KacAAR H3.3 27Kme1SAPSTGGV36Kme3KPHR H4 20Kme1VLR H3.1/3.2 27Kme0SAPATGGV36Kme3KPHR H3 9KacSTGG14KacAPR H4 GLG12KacGGA16KacR H4 GGA16KacR H3.1/3.2


H3.1/3.2 27KacSAPATGGV36Kme3KPHR H3 9Kme3STGG14KacAPR H3 9Kme2STGG14KacAPR H3 9Kme1STGG14KacAPR H4 20Kme3VLR H3 T4Kme3QTAR H3 T4Kme2QTAR H3.1/3.2


H3.1/3.2


H3.3

27K 36 me2SAPSTGGV Kme2KPHR

H3.3

27K SAPSTGGV36K ac me2KPHR

H3.1/3.2

27K SAPATGGV36K ac me1KPHR

H3.1/3.2


H3.1/3.2


H3.1/3.2 27Kme0SAPATGGV36Kme2KPHR H3.3 SAPSTGGV36Kme3KPHR

+ IS

- IS

4-10D

B

Ctl

Vitamin C 1-10D

K36me3 K27me3

+ IS

H3.1/3.2

- IS

H3.3


27K 36 me3SAPSTGGV Kme2KPHR

H3.3 27Kme2SAPSTGGV36Kme3KPHR H3.1/3.2 SAPATGGV36Kme3KPHR

1-10D

C

4-10D

H3K36me3/H3 H3K27me3/H3

1.2 **

1

Fold Change

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 30

**

** ** *

0.8

*

0.6 0.4 0.2

H3

0 Ctrl

Vc 1-10D Vc 4-10D

hNSCs

28


Page 29 of 30

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Quantification of Histone Modifications by Parallel-Reaction Monitoring

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