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Characterization of Chlamydomonas reinhardtii Core Histones by Top-Down Mass Spectrometry Reveals Unique Algaespecific Variants and Post-translational Modifications Aliyya Khan, Carlo K. Eikani, Hana Khan, Anthony T. Iavarone, and James J. Pesavento J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00780 • Publication Date (Web): 03 Dec 2017 Downloaded from http://pubs.acs.org on December 5, 2017

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Journal of Proteome Research

Characterization of Chlamydomonas reinhardtii Core Histones by Top-Down Mass Spectrometry Reveals Unique Algae-specific Variants and Post-translational Modifications

Aliyya Khan†,§, Carlo K. Eikani†,§, Hana Khan§, Anthony T. Iavarone‡

and James J. Pesavento§*

† These authors contributed equally to this work. § Department of Biology, Saint Mary’s College of California, Moraga, CA 94575 ‡ QB3/Chemistry Mass Spectrometry Facility, University of California, Berkeley, CA 94720 * Corresponding author Tel: 925-631-4430 Email: [email protected]

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Keywords: microalgae, histones, post-translational modifications, top-down mass spectrometry, epigenetics

Abstract

The unicellular microalga Chlamydomonas reinhardtii has played an instrumental role in the development of many new fields (bio-products, biofuels, etc.) as well as the advancement of basic science (photosynthetic apparati, flagellar function, etc.). Chlamydomonas’ versatility ultimately derives from the genes encoded in its genome and the way that the expression of these genes is regulated, which is largely influenced by a family of DNA binding proteins called histones. In this study, we characterize C. reinhardtii core histones, both variants and their posttranslational modifications, by chromatographic separation followed by top-down mass spectrometry (TDMS). As TDMS has not been previously used to study Chlamydomonas proteins, we show rampant artifactual protein oxidation using established nuclei purification and histone extraction methods. After addressing oxidation, both histones H3 and H4 are found to each have a single polypeptide sequence that is minimally acetylated and methylated. Surprisingly, we uncover a novel monomethylation at lysine 79 on histone H4 present on all observed molecules. Histone H2B and H2A are found to have two and three variants, respectively, and both are minimally modified. This study provides an updated assessment of the core histone proteins in the green alga C. reinhardtii by top-down mass spectrometry, and lays the foundation for further investigation of these essential proteins.

Introduction

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The industrialized world currently satisfies its energy needs by relying heavily on combustion of hydrocarbons, resulting in elevated levels of the greenhouse gas carbon dioxide. Over the course of the last 70 years, the atmospheric levels of carbon dioxide have increased by nearly 100 parts-per-million, and this is widely suspected as the main culprit in the increase of ocean and air temperatures1. In order to avoid further increases in anthropogenic carbon dioxide emissions, scientists have investigated means to generate power from renewable, emissionless sources (e.g., solar, wind, etc.) and also ways to sequester carbon dioxide from sources that generate it (e.g., coal-burning power plants). One possible way to sequester carbon dioxide is through the utilization of microalgae. While several species of microalgae have been investigated for their intrinsic ability to produce biofuels, no one species has been found that can produce the quantity of biofuel needed to satisfy our current energy needs at an economical level2. To address this issue, scientists are focusing more on genetic manipulation of microalgae to improve their efficiency in converting carbon dioxide into biomass (i.e., lipids and carbohydrates)3. To date, the microalgae most amenable to genetic manipulation is the model organism Chlamydomonas reinhardtii, as its genome has been sequenced and a significant number of genetic tools are available for this organism, including RNAi gene silencing and implementation of the CRISPR/Cas9 system for genome editing4–6. Indeed, some of these tools have already been used to determine key enzymes responsible for the generation of triacylglycerols, a key storage lipid7. In addition to genetic manipulation tools, the engineering of microalgae to produce economically viable biofuel requires a deep understanding at the genetic and epigenetic level. There are few researchers studying epigenetic changes in microalgae and thus our collective knowledge on this subject is limited. Chromatin organization, which, among other things, affects gene expression and genome stability, arises from intimate interactions between DNA and a 3



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class of small proteins called histones. Histones may alter DNA structure directly through protein:DNA electrostatic interactions or indirectly by serving as a binding platform for other chromatin-modifying proteins. In either case, the histone proteins carry out their cellular functions by presenting a specific set of chemical modifications (e.g., acetylation, methylation, phosphorylation, etc.) located at specific amino acids (e.g. lysine 9 of histone H3) near the termini of the protein. By presenting a specific set of modifications, there may be a “histone code” governing many genetic functions including turning on/off gene expression, compacting DNA into higher order structures, or even flagging a damaged region of DNA for repair8. Intact protein mass spectrometry (MS), or ‘top-down’ MS (TDMS), is quickly becoming the gold standard for protein identification, characterization and quantitation9,10. TDMS is particularly well-suited for characterizing histone proteins as it identifies the type(s) and location(s) of post-translational modification(s) (PTMs) and most importantly the combination of modifications (i.e., “code”) on these heavily modified proteins. While top-down analysis of histone proteins has been successfully applied to humans and other organisms, it has not been used to study microalgal histones. Therefore, we chose to first identify histone variants present in asynchronously grown C. reinhardtii by liquid chromatography coupled online with TDMS. We were able to distinguish closely related, unreported variants (e.g., H2A gene products of HTA2 and ch2a-IV) and novel algal-specific modifications (e.g., robust histone H4 Lys79 monomethylation). During our analysis, we serendipitously uncovered evidence of significant artifactual protein oxidation using established algal nuclei isolation and histone extraction procedures. Once the oxidation issues were corrected, we were able to identify all 4 core histones in Chlamydomonas reinhardtii resulting in 12 proteoforms characterized, including previously undescribed variants and post-translational modifications.

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Methods Cell culture and growth conditions. Two cell wall-less strains of Chlamydomonas reinhardtii (cc503 and cc-124) were obtained from the Chlamydomonas Resource Center at the University of Minnesota and used throughout this study. Algae were cultured in a 2 L Erlenmeyer flask with tris-acetate-phosphate (TAP) media at pH 7.0, horizontally rotated at 110 rpm, and grown under fluorescent lights4. These cultures were seeded at 6.5x105 cells/mL and typically grown until a density of ~4x106 cells/mL was reached. The cells were harvested through centrifugation at 2500 X g for 10 min at 4 °C in a fixed-angle rotor and algal pellets were then stored at -80 °C until processed.

Isolation of Chlamydomonas Nuclei. Nuclei were isolated from Chlamydomonas cell pellets by a modified protocol based off the work by Winck et al.11. Briefly, a cell pellet (~109 cells) was resuspended in 2-3 mL of nuclei isolation buffer (NIB)12 containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM dithiothreitol (DTT), 10 mM sodium butyrate and 1x protease inhibitor cocktail (PIC; complete, without EDTA (Roche)). NIB with the aforementioned inhibitors and reducing agent is referred to as NIBA. The resuspended cell pellet was pipetted drop-wise to a liquid nitrogen-cooled mortar and then ground to a fine, green powder. The sample was transferred to a 50 mL conical and then filled with 20 mL NIBA, placed on ice for 10 min. The cells were then centrifuged at 1260 X g for 30 min at 4 °C. The supernatant was removed and the cell pellet was resuspended in 20 mL NIBA + 1% Triton X-100 and mechanically lysed by Pasteur pipetting ~20-30 times. The sample was placed on ice for 10 min and centrifuged at 1000 X g for 30 min at 4 °C to collect nuclei. The supernatant was removed and mechanical lysis with NIBA + 1% Triton X-100 was repeated once again to ensure complete lysis of the plasma membrane. The nuclei pellet was transferred to an Eppendorf microcentrifuge tube, washed 5



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twice with 1 mL NIBA to remove the detergent, centrifuged at 1000 X g for 10 min at 4 °C, and the nuclei pellet stored at -80 °C.

Histone Extraction. Histones were extracted from the isolated nuclei pellet using a high-salt buffer and procedure adapted from Morris et al13. Briefly, nuclei isolated from ~109 cells were resuspended with 2 mL of 2 M CaCl2, 10 mM HEPES pH 7, 10 mM sodium butyrate, 1 mM PMSF, 5 mM DTT, and 1xPIC. These samples were rotated end-over-end for 1 hr at 4 °C, followed by acidification with 0.3 M HCl for 15 min on ice. The insoluble material was removed through centrifugation at 12,000 X g for 5 min at 4 °C. The supernatant, containing high-salt and acidsoluble histones, was treated with trichloroacetic acid (TCA) to a final concentration of 20% and allowed to stand on ice for 1 hr to precipitate proteins. The precipitated proteins were pelleted by centrifugation at 14,000 X g for 5 min, washed with ice-cold 20% TCA to remove residual salt, and again pelleted by centrifugation. This pellet was washed once with ice-cold acetone supplemented with ~0.1% HCl, and twice with ice-cold 100% acetone. The pellet was left to air dry for 10 minutes to remove most of the acetone. The pellet was then resuspended by adding 200 µL ddH2O, mashed with a glass pestle to break apart the pellet, and then centrifuged to removed insoluble material. The resulting histone-containing supernatant was then stored at at -20 °C.

Reverse-phase high-performance liquid chromatography (RP-HPLC) fractionation of extracted C. reinhardtii histones. Typically, 50 µg of extracted algal histones (either with or without intentional oxidation14) was injected onto a Vydac C18 column (length: 250 mm; inner diameter: 4.6 mm; particle size: 5 µm) and then subjected to HPLC separation using an Agilent 1100 system. A multi-step gradient using buffer A (0.1% trifluoroacetic acid (TFA), 5% HPLC-grade 6


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acetonitrile (ACN)) and buffer B (0.1% TFA, 95% ACN) in which the concentration of buffer B rose from 27.5% to 60% by 0.5% per min routinely separated most of histone polypeptides. Fractions were pooled and then dried by centrifugation under vacuum in a Speed-Vac. Fractions were then resuspended in ddH2O and analyzed by (SDS)-polyacrylamide gel electrophoresis (PAGE) to assess purity and amount.

SDS-polyacrylamide gel electrophoresis (PAGE) analysis of histone proteins. Extracted histones were analyzed by SDS-PAGE as originated by Laemmli15. A 15% polyacrylamide gel was run at 180 V for 60 min and then stained with 1% coomassie brilliant blue. Molecular weight standards (Bullseye pre-stained protein ladder, Midsci) and calf thymus histones (Sigma) were used to identify the migration of extracted algal histones.

Mass Spectrometry. Histone extract samples were analyzed using an LTQ-Orbitrap-XL mass spectrometer equipped with an electrospray ionization (ESI) source (Thermo Fisher Scientific, Waltham, MA). For direct infusion measurements, samples were infused from a 50-µL syringe (Hamilton, Reno, NV) using the built-in syringe pump of the LTQ-Orbitrap-XL, at a flow rate of 3 µL/min. For liquid chromatography-mass spectrometry (LC-MS) measurements, samples were analyzed using a 1200 series liquid chromatograph (Agilent, Santa Clara, CA) that was connected in-line with the mass spectrometer. The LC was equipped with a C18 analytical column (length: 50 mm; inner diameter: 1.0 mm; particle size: 5 µm; Viva C18, Restek, Bellefonte, PA) and a 100µL sample loop. Acetonitrile, formic acid (Optima grade, 99.9%, Fisher), and water purified to a resistivity of 18.2 MΩ·cm (at 25 °C) using a Milli-Q Gradient ultrapure water purification system (Millipore, Billerica, MA) were used to prepare mobile phase solvents. Solvent A was 99.9% water/0.1% formic acid and solvent B was 99.9% acetonitrile/0.1% formic acid (v/v). The elution 7



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program consisted of isocratic flow at 1% B for 5 min, a linear gradient to 20% B over 0.1 min, a linear gradient to 60% B over 34.9 min, a linear gradient to 95% B over 0.1 min, isocratic flow at 95% B for 4.9 min, a linear gradient to 1% B over 0.1 min, and isocratic flow at 1% B for 19.9 min, at a flow rate of 200 µL/min. The column compartment was maintained at 40 °C. Full-scan mass spectra were acquired in the positive ion mode over the range m/z = 500 to 1600 using the Orbitrap mass analyzer, in profile format, with a mass resolution setting of 100,000 (at m/z = 400, measured at full width at half-maximum peak height, FWHM). For tandem mass spectrometry (MS/MS) experiments, mass-selected precursor ions were fragmented using collision-induced dissociation (CID) or electron transfer dissociation (ETD), and MS/MS spectra were recorded using the Orbitrap mass analyzer, in profile format, with a mass resolution setting of 100,000 (at m/z = 400, FWHM). CID was performed under the following conditions: isolation width: 2 m/z units, normalized collision energy: 28%, default charge state: 3+, activation Q: 0.25, and activation time: 30 ms. The activation time for ETD was 5 ms. Data acquisition and analysis were performed using Xcalibur (version 2.0.7, Thermo), ProSight PTM and ProSight Lite16 software (Kelleher Research Group, Northwestern University, Evanston, IL), and MASH Suite Pro17 (Ge Research Group, University of Wisconsin, Madison, WI).

Results and Discussion Liquid Chromatography Followed by Tandem Mass Spectrometry (MS/MS) Identifies PostTranslationally Modified Histone Variants from Asynchronous C. reinhardtii Work done on C. reinhardtii by Morris and Waterborg in the early 1990s identified several histone variants and many PTMs such as methylation, acetylation and ubiquitylation13,18. The identification relied heavily on the available methods of the day, including proteolysis followed by Edman degradation for protein composition and radiolabeling for PTM localization 8


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and quantitation. Recent advances in top-down mass spectrometry (TDMS) provide a more complete approach at characterizing histone variants and their modifications. The ability to isolate a single molecular species on an intact protein inherently generates an unambiguous identification of the protein and localizes any PTMs present (unlike Edman degradation and MSbased approaches employing the use of proteases). Because intact protein mass spectrometry has not previously been performed on algal histones, we wanted to first assess the types of histone polypeptides and their proteoforms19 by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) (Figure 1). The following four core histones were identified from MS and MS/MS measurements, and eluted in an order consistent with that observed by other groups,18 namely, H2B, H4, H2A and lastly H3. A general description both of protein variants and their PTMs will be provided in this section and will be further elaborated upon in following sections. All histone variants and modified forms identified by MS and MS/MS are listed in Table 1. The first proteins to elute during RPLC were determined to be two variants of H2B, gene products of HTB4 (relative molecular mass (Mr) = 16520.1 Da, UniProt # A8HV98) and HBV1 (Mr = 13247.2 Da, UniProt # A8JCT1; Figure 1A). Both proteins were found to have α-amino acetylation on the N-terminal alanine residue (Nα-ac). Additionally, both H2B polypeptides were found to have all three internal methionine residues partially oxidized to methionine sulfoxide. The next histone to elute was identified as H4 (Mr = 11391.3 Da, UniProt # P50566), present as one polypeptide sequence with the most abundant form having an aαS1, methionine sulfoxide and a monomethylation (Figure 1B and vide infra). Another, less abundant H4 form contained the aforementioned modifications in addition to an internal lysine acetylation (Mr = 11433.4 Da). Three H2A polypeptides encoded by genes HTA10 (Mr = 13743.6 Da, Uni Prot # A8HWF6), HTA2 (Mr = 13530.6 Da, UniProt # A8HSB5), and ch2a-IV (Mr = 13587.6 Da, UniProt # Q42680) eluted 9



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shortly after H4 (Figure 1C). All three H2A variants contained a single acetylation on their Nterminal residue. Histone H3, encoded by the gene ch3-II (most abundant two masses of Mr = 15199.6 Da and 15215.5, UniProt # Q6LCW8), eluted last and exhibited a complex mass profile with ions consistent with multiple states of methylation, acetylation and oxidation. Unfortunately, the histone H3 ions exhibited poor signal-to-noise ratios (S/N), hindering our efforts to characterize the protein by MS/MS (Figure 1D).

Figure 1. LC-MS/MS analysis of salt extracts from C. reinhardtii nuclei led to the identification of the four core histones. Histone H2B (A), H4 (B), H2A (C), and H3 (D) were found to have retention times of 15, 18, 23 and 24 minutes, respectively. Masses that were targeted for MS/MS are noted by the monoisotopic mass, followed by the mass difference (∆m) from the 10


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theoretical, unmodified mass, and finally the corresponding gene name associated with the protein identified. The charge state(s) shown for H2B are +22 and +17 (A, left and right, respectively); H4 is +12; H2A is +14; H3 is +18.

Chlamydomonas histones H2A and H2B have few variants and minimal post-translational modifications. We were interested in the question of whether C. reinhardtii histone H2A and H2B variants and their PTM profiles align more closely with those of simple, unicellular organisms or more complex, multicellular organisms. Chlamydomonas has a sizable genome (121 Mb; ~15,000 genes) with a large number of genes relative to other unicellular eukaryotes (for yeast, 12.1 Mb; ~6,300 genes). In fact, it is more similar to D. melanogaster (175 Mb; ~13,600 genes) and A. thaliana (157 Mb; ~25,500 genes) than S. cerevisiae when it comes to genome size and gene number4. However, like yeast, Chlamydomonas is unicellular and thus does not have the complexity of multiple cell types found in flies, land plants or humans. TDMS analysis of C. reinhardtii H2B revealed the presence of two protein variants, with the molecular mass of the most abundant proteoform of each being 13247.2 Da (HBV1) and 16520.1 Da (HTB4), respectively (Figure 1A). Both proteoforms were found to have all internal methionine residues oxidized to the sulfoxide. Oxidation of H2B has been observed by others18,20,21, but at dramatically lower levels than those reported here (Figure 1A and Table 1). The larger H2B variant has an extended N-terminal tail of 29 amino acids that is absent in the smaller variant (Supplemental Figure 1). Interestingly, all H2B variants in A. thaliana have molecular masses of approximately 16 kDa (closest homology with Chlamydomonas H2B variant histone HTB9) as well as an extended H2B N-terminal tail. However, unlike Chlamydomonas, A. thaliana has at 11



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least five known H2B variants22, which is more along the lines of higher eukaryotes. For example, the compilation of human histone H2B variants consists of at least ten distinct sequences, and of those only one is monoacetylated (albeit minimally)23,24. Unlike algae and land plants, all human H2B variants lack an extended N-terminal sequence and have molecular masses of ~13.8 kDa25 (Supplemental Figure 2). TDMS analysis of yeast histones also revealed that, like Chlamydomonas, only two H2B variants are expressed. Different from Chlamydomonas, both yeast variants are highly acetylated26 (up to 5 internal acetylations). It has been reported that H2B variant HTB4 is highly monoubiquitylated in Chlamydomonas18. We were able to confirm the presence of this proteoform by SDS-PAGE (data not shown), but did not detect it by LC-MS/MS or direct-injection ESI-MS of offline-purified RPLC fractions (vide infra). It may be that our measurements were limited by the protein’s abundance, size (~22 kDa), or ionization efficiency. Nevertheless, the analysis of microalgal histone H2B reveals that it exhibits characteristics of both unicellular (low number of variants) and multicellular (reduced acetylation) organisms as well as plants (extended N-terminus) and animals (shortened Nterminus).

Table 1. Identification of the most abundant, intact Chlamydomonas core histone proteoforms.

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Histone

Gene

Observed Mass (Da)

H2B

HBV1

13247.2

∆m

Modification Acetylation (Nα-ac)

+90 Da 3x Oxidation (M55ox, M58ox, M69ox)

Acetylation (Nα-ac) H2B

HTB4

16520.1

+74 Da 2x Oxidation (M87ox, M90ox)

Acetylation (Nα-ac) H4

HFO4

11375.5

+56 Da Methylation (K79me1)


HFO4

11391.5

+72 Da

Methylation (K79me1) Oxidation (M84ox)


HFO4

11433.4

+98 Da

Acetylation (K5 – K16) Methylation (K79me1)

H2A

HTA2

13530.6

+42 Da

H2A

HTA2

13514.7

+26 Da

Acetylation (Nα-ac) Acetylation (Nα-ac) S9 dehydration/reduction (-16 Da)? (A1-G10)

H2A

HTA10

13743.6

+42 Da

Acetylation (Nα-ac)

H2A

ch2a-IV

13587.6

+42 Da

Acetylation (Nα-ac)

H3

ch3-II

15249.4

+80 Da

5x Oxidation (C109ox3, M119ox2)

H3

ch3-II

15263.4

+94 Da

Methylation (K4me1) Oxidation (C109ox3, M119ox2)

2x Methylation (K4me1, K27me1) H3

ch3-II

15277.4

+108 Da 5x Oxidation (C109ox3, M119ox2)

All core histones were found to be lacking an N-terminal methionine, therefore amino acid numbering starts at the first amino acid present in the sequence. Modifications that could not be localized to a single amino acid are indicated by bold font.

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The three H2A variants retain complete amino acid sequence identity until the very last ~5 – 8 C-terminal amino acids (Supplemental Figure 1). In his seminal paper first characterizing Chlamydomonas histones18, Waterborg named histone H2A variants based on electrophoretic mobility, but could not differentiate the HTA2 from the ch2a-IV gene product due to their similar molecular masses (unmodified Mr of 13488.6 Da and 13545.6 Da, respectively). In keeping with his nomenclature, we propose to keep the original name of H2A.1 for the HTA10 gene product with the largest molecular mass (unmodified Mr of 13701.71 Da) and name the ch2a-IV and HTA2 gene product H2A.2 and H2A.3, respectively, based on decreasing molecular masses. For all three H2A variants, a minor species with a molecular mass lower by 16 Da than the mass of the most abundant species was observed (26 Da = 42 Da – 16 Da; Figure 1C). Despite its low abundance, we were able to obtain MS/MS measurements of this precursor ion of the H2A.3 (13514.7 in Figure 1C) and localize the -16-Da mass shift to the first 10 amino acids of H2A.3 (data not shown). Since all three H2A variants share the identical amino acid sequence until the 124th amino acid residue and all three forms show a -16-Da shoulder (Figure 1C), we suspect that this -16-Da PTM is also localized to the N-terminal region of H2A.1 and H2A.2. We believe this is an in vivo modification since no other histones show a loss of 16 Da from the theoretical unmodified mass. Additionally, we performed experiments to determine whether this -16-Da species was due to procedural handling: Calf thymus histone that was spiked into the algal histone preparation (vide infra) did not show a loss of 16 Da on calf H2A, while this loss was readily detectable on algal H2A within the same LC-MS/MS experiment (data not shown). One possible PTM that could account for the 16-Da decrease in molecular mass is dehydration followed by reduction at serine 9; however, further investigation is required for confirmation.

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Tandem MS Analysis of Chlamydomonas Histone H4 Reveals Extensive Monomethylation at Lysine 79 Typically, LC-MS analyses of C. reinhardtii histone extracts show a histone H4 MS profile with two major forms: one with an Mr of 11391.3 Da and a ∆m of +72 Da (~85% of total H4 protein) and one with an Mr of 11433.4 Da and a ∆m of +114 Da (~15% of total H4 protein) (Figure 1B). Based on the mass difference alone, the +72-Da species could be accounted for by a combination of acetylation (+42 Da), monomethylation (+14 Da), and oxidation (+16 Da). Likewise, the +114-Da form could have all of the aforementioned modifications as well as an additional acetylation (72 Da + 42 Da = 114 Da). However, depending on the sample, varying degrees of unintentional oxidation can confound MS and MS/MS analysis (for example, compare Figure 1B with Figure 2A). Despite the complications, we were able to completely characterize the PTMs on Chlamydomonas histone H4 (Table 1). A data-directed MS/MS approach targeting multiple charge states of the 11375.5-Da species for collision-induced dissociation (CID) generated enough protein sequence coverage to conclusively localize the mass differences to an N-terminal acetylation at serine 1 (+42 Da) and a monomethylation at lysine 79 (+14 Da) (Figure 2B). The +14-Da mass increase was localized to K79 or T80 by using the y222+ ion (∆m = 0 Da) and the y242+ ion (∆m = +14 Da), which suggested the presence of monomethylation on K79 (Figure 2B). The existence of H4K79me1 was corroborated by subsequent ETD fragmentation of the 11375.5-Da species (data not shown). CID fragmentation of the 11391.5-Da (∆m = +72 Da) species yielded fewer and less abundant ions compared to the unoxidized, 11375.5-Da (∆m = +56 Da) H4 form. A significant number of fragment ions from this oxidized form were found to have a 64-Da loss (i.e., methanesulfenic acid), due to the labile side chain of methionine sulfoxide27, and could not localize the monomethylation to a single amino acid residue. However, we performed ETD (i.e., gentler 15



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fragmentation than CID) of the oxidized, monomethylated form (Mr = 11391.5 Da) and identified two conclusive c ions (c78 and c79) that indeed confirmed H4K79 is monomethylated (Figure 2C).

Figure 2. MS and MS/MS measurements of histone H4 from C. reinhardtii reveal extensive monomethylation at lysine 79. (A) ESI mass spectrum of intact histone H4 (14+ charge state) shows two predominant masses. (B) Graphical fragmentation map showing results from CID of the 11375.4-Da species (N-terminal acetylation in red, K79 monomethylation in green). Two key fragment ions, y22 and y24, localize the monomethylation to K79 (dotted box in graphical representation). (C) Graphical fragmentation map showing results from ETD of the 11391.4-Da species (N-terminal acetylation in red, K79 monomethylation in green, M84 sulfoxide in grey). 16


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Three key fragment ions (c77, c78 and c79) localize the monomethylation to K79 (dotted box in graphical representation).

The fact that nearly 100% of H4K79 was found to be monomethylated in Chlamydomonas was very surprising, as this modification has not been reported for any organism when TDMS was used as the analytical approach. Even when the inherent dynamic range limitation of TDMS on RPLC-purified proteins was mitigated by use of additional chromatographic separation (detection limit of ~0.001% of total protein28), H4K79 monomethylation was not detected in HeLa (human) cells. However, there have been reports using bottom-up MS that estimate H4K79 monomethylation in HeLa cells to be anywhere from

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