Assessment of Quantification Precision of Histone Post-Translational

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Assessment of quantification precision of histone post-translational modifications by using an ion trap and down to 50,000 cells as starting material Qi Guo, Simone Sidoli, Benjamin A. Garcia, and Xiaolu Zhao J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00544 • Publication Date (Web): 09 Nov 2017 Downloaded from http://pubs.acs.org on November 10, 2017

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Journal of Proteome Research

Assessment of quantification precision of histone post-translational modifications by using an ion trap and down to 50,000 cells as starting material 1

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1,

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Qi Guo , Simone Sidoli , Benjamin A. Garcia * and Xiaolu Zhao * 1

Epigenetics Institute, Department of Biochemistry and Biophysics, Perelman School of Medicine,

University of Pennsylvania, Philadelphia, PA 19104, USA 2

Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan,

P.R.China, 430072

*Corresponding authors: Benjamin A. Garcia, Epigenetics Institute, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Room 9-124, 3400 Civic Center Blvd, Bldg 421, Philadelphia, PA 19104, USA. E-mail: [email protected]; Phone: 1-215-573-9423; Fax: 215-573-4764 Xiaolu Zhao, Room 6016, College of Life Sciences, Wuhan University, Luojiashan Road, Wuhan, P.R.China, 430072. Email: [email protected]; Tel: + 86-27-68753797: Fax: + 86-27-68753797

Keywords: data independent acquisition, histones, mass spectrometry, post-translational modifications, bottom-up

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Abstract Histone post-translational modifications (PTMs) are fundamental players of chromatin regulation, as they contribute to editing histone chemical properties and recruiting proteins for gene transcription and DNA repair. Mass spectrometry (MS) based proteomics is currently the most widely adopted strategy for high throughput quantification of hundreds of histone PTMs. Samples such as primary tissues, complex model systems and biofluids are hard to retrieve in large quantities. Because of this, it is critical to know whether the amount of sample available would lead to an exhaustive analysis if subjected to MS. In this work, we assessed the reproducibility in quantification of histone PTMs using a wide range of starting material, i.e. from 5,000,000 to 50,000 cells. We performed the experiment using four different cell lines, i.e. HeLa, 293T, human embryonic stem cells (hESCs) and myoblasts, and we quantified a list of 205 histone peptides using ion trap MS and our in-house software. Results highlighted that the relative abundance of some histone PTMs deviated as little as just 4% when comparing high starting material with histone samples extracted from 50,000 cells, e.g. H3K9me2 (40% average abundance). Low abundance PTMs such as H3K4me2 (10Hz), high resolution (>100,000) and high sensitivity of MS made it suitable for online chromatographic separation and detection of total modified histone 6

peptides within an hour . Histones can be analyzed with the traditional bottom-up MS strategy, and also via middle-down or top-down MS (reviewed in 7), in order to accurately identify and quantify not only individual PTMs, but also their co-frequency. The most commonly used strategy is still bottom-up MS, and the most widely adopted protocol includes derivatization of lysine residues in histones to allow trypsin to generate Arg-C like peptides (4-20 aa)

8-10

. Data-independent acquisition (DIA) is

currently the most suitable MS acquisition method, due to the large variety of isobarically modified peptides, which require MS/MS based quantification to discriminate their abundance if co-eluting 11-13

during chromatography

. Recently, Sidoli et al. assessed that the relatively low complexity of



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purified histone samples combined with DIA allows for the use of low resolution MS instrumentation 12

such as the ion trap , paving the way for a more affordable analysis of histone PTMs. Since biological material is not always available in large quantities, it is important to assess the amount required for specific experiments. For example, primary cells are usually more biologically relevant tools than cell lines for biological studies; however, obtaining a pure population of primary cells can be a difficult and arduous process due to their requirement of additional nutrients not included in classical media. The amount of material required has been established for several biochemical techniques, including large-scale proteomics, i.e. 1-2 µg of peptides on column when running nano liquid chromatography (nanoLC). Histones is a peculiar example, as they are among the most abundant proteins in eukaryotic cells. Considering the length of the human genome (3 billion base pairs) and the average distribution of nucleosomes (about one each ~200 base pairs), it is safe to assume that histone proteins are present in millions and millions of copies in each individual cell. Because of this, a low cell number might be sufficient to perform exhaustive histone PTM analysis, but this still needed to be assessed. In this work, we demonstrate that histone PTMs can be characterized using low resolution MS (ion trap) starting with as low as 50,000 cells. To put that in context, this cell number can be easily grown in a well of a 96-well plate when using average suspension cells. We assessed the reliability of quantification with such small amount of cells by using a variety of cell lines with remarkably different phenotypes: Hela, 293T, human embryonic stem cells (hESC) and myoblasts. In the low cell number range, we established that histone marks with low abundance such as H3K4me had unfavorable coefficient of variation, and thus we suggest using more cells when characterizing those low abundance histone marks. However, abundant histone marks such as histone H4 acetylations were efficiently quantified with low cell counts. Collectively, our study addresses a simple, albeit critical, question about the amount of material required for MS analysis of histone PTMs. Moreover, the study was performed with low resolution MS, showing that this analysis is possible with cost efficient instrumentation.


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Materials and Method Cell culture – HeLa cell line (CCL-2) was cultured at 37°C with 5% CO2 in Dulbecco’s Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, 2 mM L-glutamine and 50 μg/ml each of penicillin/streptomycin. Cultures at about 80% confluence were split 1:5 in 10 cm culture dishes. Briefly, the cells were washed in PBS. PBS containing 0.25% (w/v) trypsin was added to the dishes and placed at 37°C for 5-10 minutes. After the cells were detached from the dishes, prewarmed culture medium was added and the cells were pipetted up and down to render them single cells. Primary myoblasts were cultured on dishes coated with 0.1% gelatin (Millipore) as described 14

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previously . Briefly, cells were grown at 2000 to 3000 cells/cm in proliferation medium composed of Dulbecco modified Eagle medium (DMEM), Medium 199 (Hyclone), 15% FBS, (Hyclone), 0.02 M HEPES buffer, 1.4 mg/l vitamin B12 (both Sigma-Aldrich, St. Louis, MO, USA), 0.03 mg/l ZnSO4 (Fisher Scientific, Fair Lawn, NJ, USA), 0.055 mg/l dexamethasone (Sigma-Aldrich), 2.5 μg/l hepatocyte growth factor and 10 μg/l basic fibroblast growth factor (both from Biovision Inc). At 80% confluency (usually every 5 to 7 days), cells were detached using 0.05% trypsin/EDTA (Gibco), quenched with DMEM/10% FBS dispersed into single cells and counted. H9 hESC from WiCell (Madison, WI) were maintained on Matrigel (Corning/Stem Cell Technologies) coated 6-well tissue culture plates in mTeSR®1 medium (Stem Cell Technologies, Vancouver, BC, Canada) as per manufacturer’s recommendation. Medium was changed daily and cells were used when 60–80% confluent. Colonies were enzymatically detached using Accutase (eBioscience, San Diego, CA) and dispersed by pipetting for cell counting. 293T cells were grown adherent on plates with DMEM containing 10% FBS, 50 μg/ml each of penicillin and streptomycin. The cells were passaged every 3-4 days when at 80% confluency. Cells were harvested in 0.05% trypsin/EDTA and dispersed into single cells for counting. Cell numbers were determined using a hemocytometer. To aliquot different cell numbers of 5e4, 1e5, 2e5, 5e5, 1e6, 2e6 and 5e6, about 10 million cells were set aside from each cell line in triplicate, washed in PBS thoroughly and collected by centrifugation at 150xg. One biological set was used to confirm extraction of histones using PAGE. Two other replicates were analyzed for MS. Histone extraction – Histones were extracted as described previously with minor modification6. The cells were incubated in nuclear isolation buffer (NIB) (15 mM Tris–HCl, 15 mM NaCl, 60 mM KCl, 5



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mM MgCl2, 1 mM CaCl2, 250 mM sucrose, pH 7.5, and 0.5 mM AEBSF, 10 mM sodium butyrate, 5 nM microcystein, 1 mM DTT added fresh) with 0.3% NP-40 on ice for 5 min. 300 μl, 200 μl, 100 μl, 100 μl, 50 μl, 50 μl and 50 μl of NIB with NP-40 were used for 5e6, 2e6, 1e6, 5e5, 2e5, 1e5 and 5e4 cells. The nuclei were collected by centrifuging at 700 × g at 4°C for 5 min. The resulting nuclear pellet was washed twice with the same volume of nuclear isolation buffer without NP-40. Histones were then acid-extracted with 0.2 M H2SO4 for 3 hours at 4°C with rotation. For all cells numbers, 100 μl of 0.2 M H2SO4 were used. The insoluble nuclear debris were pelleted at 3400 × g at 4°C for 5 min, and the supernatant was retained. Finally, histone proteins were precipitated overnight on ice after adding 100% trichloroacetic acid (TCA) in the ratio of 1:3 (v/v) to the acid extraction supernatant, in order to obtain a final TCA concentration of 33%. The precipitated proteins were washed once with 0.1% HCl in acetone (−20°C), twice with acetone (−20°C), and dried in speedvac. One biological set was redissolved in 10 μl of ddH20. Protein concentration was measured using the Bradford assay and histone proteins were subjected to 15% SDS-PAGE. Two other replicates were redissolved in 30 μl of 50 mM NH4HCO3 (pH 8.0) and subjected to histone propionylation and digestion. Histone propionylation and digestion - The histone propionylation and digestion were 6

performed as previously described with minor modification . Propionic anhydride solution was freshly prepared by mixing propionic anhydride with acetonitrile in a ratio of 1:3 (v/v). 15 μl of such derivatization reagent was mixed with the histone sample in the ratio of 1:2 (v/v) and 7.5 μl of ammonium hydroxide was added immediately to the mixture, followed by an incubation of 15 minutes at 37°C. After incubation, the samples were dried in a speed-vac to

Assessment of Quantification Precision of Histone Post-Translational

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