Middle‐Down and Top‐Down Mass Spectrometric Analysis of Co‐occurring Histone Modifications

Rosalynn C. Molden1, Benjamin A. Garcia2

1 Department of Chemistry, Frick Laboratory, Princeton University, Princeton, New Jersey, 2 Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 23.7
DOI:  10.1002/0471140864.ps2307s77
Online Posting Date:  August, 2014
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Abstract

Histones are chromatin proteins that are highly modified with many different types of post‐translational modifications. These modifications act in concert to regulate a number of chromatin‐related processes. However, identification and quantification of co‐occurring histone post‐translational modifications is challenging because there are many potential combinations of modifications and because the commonly used strategy of fragmenting proteins using trypsin or an alternative protease prior to LC‐MS/MS analysis results in the loss of connectivity between modifications on different peptides. In this unit, mass spectrometric methods to analyze combinatorial histone modifications on histone tails (middle‐down mass spectrometry) and on intact histones (top‐down mass spectrometry) are described. Curr. Protoc. Protein Sci. 77:23.7.1‐23.7.28. © 2014 by John Wiley & Sons, Inc.

Keywords: histone modifications; top‐down mass spectrometry; middle‐down mass spectrometry; post‐translational modifications

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Middle‐Down MS Analysis of Histone H3 Using On‐Line WCX‐HILIC
  • Basic Protocol 2: Middle‐Down MS Analysis of Histone H4 Using Off‐Line HILIC Fractionation
  • Basic Protocol 3: Top‐Down MS Analysis of Intact Histones Using ETD
  • Alternate Protocol 1: Off‐Line HILIC Separation of Intact Histones
  • Support Protocol 1: Extraction and Purification of Mammalian Histones from Cell Lines
  • Support Protocol 2: Preparation of Capillary Columns with Integrated Emitters
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Middle‐Down MS Analysis of Histone H3 Using On‐Line WCX‐HILIC

  Materials
  • Dry HPLC‐purified histone H3 variant (see protocol 5)
  • Glu‐C (see recipe)
  • 100 mM ammonium acetate, pH 4.0 (store at room temperature)
  • Off‐line reversed‐phase buffers A and B (see recipes)
  • PolyCat A resin, 300 Å, 3 µm (PolyLC, cat. no. BMCT0315)
  • On‐line WCX‐HILIC buffers A and B (see recipes)
  • Beckman Coulter HPLC, System Gold, or equivalent off‐line HPLC
  • Vydac C18 reversed‐phase column, 250 mm, 2.1 mm, 5 µm, 300 Å pore size (Grace, cat. no. 218TP52) or equivalent
  • SpeedVac evaporator
  • Sample vials for MS
  • Eksigent nanoLC‐Ultra 2D HPLC with AS2 autosampler or equivalent nanoflow system or split‐flow system
  • HILIC capillary column, 10 mm, 50 µm i.d., 360 µm o.d. (Nest Group, cat. no. P15M05CT0503)
  • LTQ Orbitrap XL with ETD (Thermo Scientific)

Basic Protocol 2: Middle‐Down MS Analysis of Histone H4 Using Off‐Line HILIC Fractionation

  Materials
  • Dry HPLC‐purified histone H4 (see protocol 5)
  • 100 mM ammonium bicarbonate, pH 8.0 (store at 4oC)
  • Asp‐N (Roche, cat. no. 11370529001)
  • Glacial acetic acid (>99.9%)
  • Trifluoroacetic acid (TFA; optional)
  • HPLC‐grade methanol
  • LC/MS‐grade water
  • LC/MS‐grade acetonitrile
  • Magic C18AQ resin, 200 Å, 3 µm (Michrom Bioresources, cat. no. PM5/61100/00)
  • On‐line reversed‐phase buffers A and B (see recipes)
  • SpeedVac evaporator
  • Pipet tip
  • Empore C8 disk, 1‐mm diameter (3M, cat. no. 2214)
  • Reversed‐phase C18 capillary column, 150 mm, 75 µm i.d., 360 µm o.d. (see protocol 6)
  • LTQ Orbitrap XL with ETD (Thermo Scientific)
  • Sample vials for MS
  • Eksigent nanoLC‐Ultra HPLC and Eksigent AS2 autosampler or equivalent nanoflow system or split‐flow system

Basic Protocol 3: Top‐Down MS Analysis of Intact Histones Using ETD

  Materials
  • Magic C18AQ resin, 5 µm, 100 Å (Michrom Bioresources, cat. no. PM5/61100/00)
  • LC/MS‐grade acetonitrile
  • Equine myoglobin (Sigma, cat. no. M0630)
  • Glacial acetic acid (>99.9%)
  • Purified histones (see protocol 5)
  • Fused‐silica capillary tubing, 20 µm i.d., 360 µm o.d. (Polymicro, cat. no. TSP0203752)
  • Infusion column, 50 µm i.d., 365 µm o.d. (see protocol 6)
  • 25‐µl Hamilton syringe (cat. no. 80275)
  • LTQ Orbitrap XL with ETD

Alternate Protocol 1: Off‐Line HILIC Separation of Intact Histones

  Materials
  • Off‐line HILIC buffers A and B (see recipes)
  • Histone sample, HPLC‐purified intact protein or H3(1‐50) peptide
  • 100% (w/v) TCA solution (see recipe)
  • 0.1% (v/v) HCl in acetone, −20°C
  • Acetone, −20°C
  • HPLC‐grade methanol
  • 0.1 M acetic acid in HPLC‐grade water
  • 0.1 M acetic acid in 75% HPLC‐grade acetonitrile/25% HPLC‐grade water
  • HPLC with PolyCAT A column, 4.6 mm, 150 mm, 3 µm, 1000 Å (PolyLC)
  • SpeedVac evaporator
  • Empore C8 (3M, cat. no. 2214)
  • Compressed air canister (optional)

Support Protocol 1: Extraction and Purification of Mammalian Histones from Cell Lines

  Materials
  • Nuclear isolation buffer (NIB; see recipe)
  • 5 M sodium butyrate (see recipe)
  • 100× HALT protease and phosphatase inhibitor cocktail, EDTA free (Thermo Pierce, cat. no. 78441)
  • 1 M DTT (prepare fresh; Invitrogen, cat. no. 15508‐013)
  • 10% NP‐40 (CalBiochem, cat. no. 492016)
  • Mammalian cells
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • 0.4 N sulfuric acid (H 2SO 4)
  • 100% (w/v) TCA solution (see recipe)
  • 0.1 % (v/v) HCl in acetone (store at −20°C)
  • Cold acetone (store at −20°C)
  • Off‐line reversed‐phase HPLC buffers A and B (see recipes)
  • Tabletop centrifuge
  • Cell rotator
  • Beckman Coulter System Gold HPLC with UV detector and ProteomeLab FC module fraction collector
  • Vydac 4.6‐mm reversed‐phase column, 5 µm, 300 Å pore size (Grace, Vydac, cat. no. 218TP54) or equivalent
  • SpeedVac evaporator

Support Protocol 2: Preparation of Capillary Columns with Integrated Emitters

  Materials
  • 50% reagent‐grade methanol in Milli‐Q water
  • HPLC‐grade isopropanol
  • HPLC‐grade acetonitrile
  • Appropriate resin for specific application
  • On‐line reversed‐phase buffers A and B (see recipes)
  • 2 ng/µl angiotensin (Sigma, cat. no. A9650) in on‐line reversed‐phase buffer A
  • Fused‐silica capillary tubing, 50 or 75 µm i.d., 360 µm o.d. (Polymicro, cat. no. BCT0303 or 1068150017)
  • Ceramic scoring wafer (Restek, cat. no. 23015)
  • Laser micropipet puller (Sutter Instrument Co., Model P‐2000)
  • Lighter
  • Microscope
  • Glass vial
  • Flea stir bar, 12 × 35 mm (Fisherbrand, cat. no. 03‐339‐25A)
  • Appropriate column for specific application
  • Pressure bomb (Nanobaume, cat. no. SP‐400), attached to pressurized gas tank (e.g., helium) with tubing and fittings capable of withstanding 9000 psig, including three‐way valve with a bottom vent to regulate gas flow to the bomb
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Figures

Videos

Literature Cited

Literature Cited
  Banerjee, T. and Chakravarti, D. 2011. A peek into the complex realm of histone phosphorylation. Mol. Cell Biol. 31:4858‐4873.
  Boyne, M.T.2nd, Pesavento, J.J., Mizzen, C.A., and Kelleher, N.L. 2006. Precise characterization of human histones in the H2A gene family by top down mass spectrometry. J. Proteome Res. 5:248‐253.
  DiMaggio, P.A., Young, N.L., Baliban, R.C., Garcia, B.A., and Floudas, C.A. 2009. A mixed integer linear optimization framework for the identification and quantification of targeted post‐translational modifications of highly modified proteins using multiplexed electron transfer dissociation tandem mass spectrometry. Mol. Cell Proteomics 8:2527‐2543.
  Eliuk, S.M., Maltby, D., Panning, B., and Burlingame, A.L. 2010. High resolution electron transfer dissociation studies of unfractionated intact histones from murine embryonic stem cells using on‐line capillary LC separation: Determination of abundant histone isoforms and post‐translational modifications. Mol. Cell Proteomics 9:824‐837.
  Fischle, W., Tseng, B.S., Dormann, H.L., Ueberheide, B.M., Garcia, B.A., Shabanowitz, J., Hunt, D.F., Funabiki, H., and Allis, C.D. 2005. Regulation of HP1‐chromatin binding by histone H3 methylation and phosphorylation. Nature 438:1116‐1122.
  Fuchs, S.M., Krajewski, K., Baker, R.W., Miller, V.L., and Strahl, B.D. 2011. Influence of combinatorial histone modifications on antibody and effector protein recognition. Curr. Biol. 21:53‐58.
  Garcia, B.A., Pesavento, J.J., Mizzen, C.A., and Kelleher, N.L. 2007. Pervasive combinatorial modification of histone H3 in human cells. Nat. Methods 4:487‐489.
  Garcia, B.A., Thomas, C.E., Kelleher, N.L., and Mizzen, C.A. 2008. Tissue‐specific expression and post‐translational modification of histone H3 variants. J. Proteome Res. 7:4225‐4236.
  Goodlett, D.R., Yi, E.C., and Mottay, P. 2007. Preparation and use of an integrated microcapillary HPLC column and ESI device for proteomic analysis. Cold Spring Harb. Protoc. 2007:pdb.prot4617.
  Guner, H., Close, P.L., Cai, W., Zhang, H., Peng, Y., Gregorich, Z.R., and Ge, Y. 2014. MASH Suite: A user‐friendly and versatile software interface for high‐resolution mass spectrometry data interpretation and visualisation. J. Am. Soc. Mass Spectrom. 25:464‐470.
  Hu, G. and Wade, P.A. 2012. NuRD and pluripotency: A complex balancing act. Cell Stem Cell 10:497‐503.
  Jung, H.R., Sidoli, S., Haldbo, S., Sprenger, R.R., Schwammle, V., Pasini, D., Helin, K., and Jensen, O.N. 2013. Precision mapping of coexisting modifications in histone H3 tails from embryonic stem cells by ETD‐MS/MS. Anal. Chem. 85:8232‐8239.
  LeDuc, R.D. and Kelleher, N.L. 2007. Using ProSight PTM and related tools for targeted protein identification and characterization with high mass accuracy tandem MS data. Curr. Protoc. Bioinform. 19:13.6.1‐13.6.28.
  Lindner, H., Sarg, B., Meraner, C., and Helliger, W. 1996. Separation of acetylated core histones by hydrophilic‐interaction liquid chromatography. J. Chromatography A 743:137‐144.
  Link, A.J. and LaBaer, J. 2009. Proteomics: A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  McAlister, G.C., Phanstiel, D., Good, D.M., Berggren, W.T., and Coon, J.J. 2007. Implementation of electron‐transfer dissociation on a hybrid linear ion trap‐orbitrap mass spectrometer. Anal. Chem. 79:3525‐3534.
  Moriniere, J., Rousseaux, S., Steuerwald, U., Soler‐Lopez, M., Curtet, S., Vitte, A.L., Govin, J., Gaucher, J., Sadoul, K., Hart, D.J., Krijgsveld, J., Khochbin, S., Muller, C.W., and Petosa, C. 2009. Cooperative binding of two acetylation marks on a histone tail by a single bromodomain. Nature 461:664‐668.
  Park, P.J. 2009. ChIP‐seq: Advantages and challenges of a maturing technology. Nat. Rev. Genet. 10:669‐680.
  Pesavento, J.J., Garcia, B.A., Streeky, J.A., Kelleher, N.L., and Mizzen, C.A. 2007. Mild performic acid oxidation enhances chromatographic and top down mass spectrometric analyses of histones. Mol. Cell Proteomics 6:1510‐1526.
  Pesavento, J.J., Bullock, C.R., LeDuc, R.D., Mizzen, C.A., and Kelleher, N.L. 2008. Combinatorial modification of human histone H4 quantitated by two‐dimensional liquid chromatography coupled with top down mass spectrometry. J. Biol. Chem. 283:14927‐14937.
  Phanstiel, D., Brumbaugh, J., Berggren, W.T., Conard, K., Feng, X., Levenstein, M.E., McAlister, G.C., Thomson, J.A., and Coon, J.J. 2008. Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 105:4093‐4098.
  Plazas‐Mayorca, M.D., Zee, B.M., Young, N.L., Fingerman, I.M., LeRoy, G., Briggs, S.D., and Garcia, B.A. 2009. One‐pot shotgun quantitative mass spectrometry characterization of histones. J. Proteome Res. 8:5367‐5374.
  Rappsilber, J., Ishihama, Y., and Mann, M. 2003. Stop and go extraction tips for matrix‐assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75:663‐670.
  Sarg, B., Helliger, W., Talasz, H., Koutzamani, E., and Lindner, H.H. 2004. Histone H4 hyperacetylation precludes histone H4 lysine 20 trimethylation. J. Biol. Chem. 279:53458‐53464.
  Sarg, B., Gréen, A., Söderkvist, P., Helliger, W., Rundquist, I., and Lindner, H.H. 2005. Characterization of sequence variations in human histone H1.2 and H1.4 subtypes. FEBS J. 272:3673‐3683.
  Schieltz, D.M., Washburn, M.P., and Hays, L.G. 2006. Analysis of complex protein mixtures using nano‐LC coupled to MS/MS. Cold Spring Harb. Protoc. 2006:pdb.prot4553.
  Shechter, D., Dormann, H.L., Allis, C.D., and Hake, S.B. 2007. Extraction, purification and analysis of histones. Nat. Protoc. 2:1445‐1457.
  Shukla, V., Vaissière, T., and Herceg, Z. 2008. Histone acetylation and chromatin signature in stem cell identity and cancer. Mutation Res. Fund. Mol. Mech. Mutagen. 637:1‐15.
  Sidoli, S., Cheng, L., and Jensen, O.N. 2012. Proteomics in chromatin biology and epigenetics: Elucidation of post‐translational modifications of histone proteins by mass spectrometry. J. Proteomics 75:3419‐3433.
  Siuti, N., Roth, M.J., Mizzen, C.A., Kelleher, N.L., and Pesavento, J.J. 2006. Gene‐specific characterization of human histone H2B by electron capture dissociation. J. Proteome Res. 5:233‐239.
  Taverna, S.D., Ilin, S., Rogers, R.S., Tanny, J.C., Lavender, H., Li, H., Baker, L., Boyle, J., Blair, L.P., Chait, B.T., Patel, D.J., Aitchison, J.D., Tackett, A.J., and Allis, C.D. 2006. Yng1 PHD finger binding to H3 trimethylated at K4 promotes NuA3 HAT activity at K14 of H3 and transcription at a subset of targeted ORFs. Mol. Cell 24:785‐796.
  Thomas, C.E., Kelleher, N.L., and Mizzen, C.A. 2006. Mass spectrometric characterization of human histone H3: A bird's eye view. J. Proteome Res. 5:240‐247.
  Tian, Z., Tolic, N., Zhao, R., Moore, R.J., Hengel, S.M., Robinson, E.W., Stenoien, D.L., Wu, S., Smith, R.D., and Pasa‐Tolic, L. 2012. Enhanced top‐down characterization of histone post‐translational modifications. Genome Biol. 13:R86.
  Wang, L.H., Li, D.Q., Fu, Y., Wang, H.P., Zhang, J.F., Yuan, Z.F., Sun, R.X., Zeng, R., He, S.M., and Gao, W. 2007. pFind 2.0: A software package for peptide and protein identification via tandem mass spectrometry. Rapid Comm. Mass Spectrom. 21:2985‐2991.
  Young, N.L., DiMaggio, P.A., Plazas‐Mayorca, M.D., Baliban, R.C., Floudas, C.A., and Garcia, B.A. 2009. High throughput characterization of combinatorial histone codes. Mol. Cell Proteomics 8:2266‐2284.
  Young, N.L., Plazas‐Mayorca, M.D., DiMaggio, P.A., Flaniken, I.Z., Beltran, A.J., Mishra, N., LeRoy, G., Floudas, C.A., and Garcia, B.A. 2010a. Collective mass spectrometry approaches reveal broad and combinatorial modification of high mobility group protein A1a. J. Am. Soc. Mass Spectrom. 21:960‐970.
  Young, N.L., Plazas‐Mayorca, M.D., and Garcia, B.A. 2010b. Systems‐wide proteomic characterization of combinatorial post‐translational modification patterns. Exp. Rev. Proteomics 7:79‐92.
  Zamdborg, L., LeDuc, R.D., Glowacz, K.J., Kim, Y.B., Viswanathan, V., Spaulding, I.T., Early, B.P., Bluhm, E.J., Babai, S., and Kelleher, N.L. 2007. ProSight PTM 2.0: Improved protein identification and characterization for top down mass spectrometry. Nucleic Acids Res. 35:W701‐W706.
Internet Resources
  http://www.actrec.gov.in/histome/index.php
  Includes sequences and modification sites for all histones and histone variants.
  http://prospector.ucsf.edu/prospector/cgi‐bin/msform.cgi?form=msproduct
  A useful tool for predicting c and z ion masses given a protein sequence. It will also match predicted ions to an imported peak list from your ETD MS2 spectrum.
  http://prosightptm2.northwestern.edu
  Can be used for database searching of ETD MS2 spectra of peptides and intact proteins.
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