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Sensitive, Specific, and Quantitative FTICR Mass Spectrometry of Combinatorial Post-Translational Modifications in Intact Histone H4 C. Logan Mackay,*,†,⊥ Bernard Ramsahoye,‡ Karl Burgess,§ Ken Cook,| Stefan Weidt,† James Creanor,† David Harrison,‡ Pat Langridge-Smith,†,⊥ Ted Hupp,‡,⊥ and Larry Hayward‡ SIRCAMS, School of Chemistry, University of Edinburgh, Edinburgh, U.K., EH9 3JJ, Institute of Genetics and Molecular Medicine, Division of Cancer Biology, University of Edinburgh, Crewe Road South, Edinburgh, U.K., EH4 2XR, Henry Welcome Functional Genomics Facility, Joseph Black Building, University of Glasgow, Glasgow, U.K., G12 8QQ, Dionex Ltd., 4 Albany Court, Albany Court Industrial Estate, Camberley, U.K., and RASOR Interdisciplinary Research Collaboration in Proteomic Technologies, University of Glasgow, Glasgow, U.K., G12 8QQ We describe a quantitative Fourier transform ion cyclotron resonance mass spectrometric (FTICR MS) analysis of the relative proportions of post-translational modification states (PTMs) of core histones in cultured cells and tissues. A novel preseparation process using a monolithic column interfaced to a 12 T FTICR MS equipped with electron capture dissociation (ECD) yields very high mass accuracy spectra, allowing direct assignment of the PTMs present in the dominant modification states of intact H4, resolving a well recognized ambiguity between trimethylation and acetylation states. By eliminating preseparation, we also obtain a highly quantitative analysis of the distribution of H4 PTMs. Rapid, extensive, and reversible effects on PTMs induced by a histone deacetylase inhibitor indicate that H4 and other core histones are accessible to modification throughout the chromatin, not just in regions of active transcription. These methods provide tools for analysis of the histone code and its role in chromatin function. In eukaryotes, nuclear DNA exists in a nucleoprotein complex called chromatin, the fundamental unit of which is the nucleosome consisting of a central histone octamer core (containing two molecules each of histone H2A, H2B, H3, and H4) around which the DNA is wrapped twice. In the nucleus, strings of nucleosomes are packaged into higher order chromatin structures regulated at several spatial scales, controlling access of enzymes involved in replication, transcription, and DNA repair.1–4 Remodeling of * Corresponding author. C. Logan Mackay, phone (0131) 651 3048, e-mail
[email protected]. † SIRCAMS, School of Chemistry, University of Edinburgh. ‡ Institute of Genetics and Molecular Medicine, Division of Cancer Biology, University of Edinburgh. § Henry Welcome Functional Genomics Facility, Joseph Black Building, University of Glasgow. | Dionex Ltd. ⊥ RASOR Interdisciplinary Research Collaboration in Proteomic Technologies, University of Glasgow. (1) Gilbert, N.; Boyle, S.; Fiegler, H.; Woodfine, K.; Carter, N. P.; Bickmore, W. A. Cell 2004, 118, 555–66. (2) Gilbert, N.; Ramsahoye, B. Brief. Funct. Genomics Proteomics 2005, 4, 129– 42. (3) Bassal, S.; El-Osta, A. Hum. Mutat. 2005, 25, 101–9. 10.1021/ac702452d CCC: $40.75 2008 American Chemical Society Published on Web 05/06/2008
chromatin is therefore required during these processes.5 The precise structural basis of chromatin remodeling remains poorly defined but the association of specific and dynamic histone posttranslational modifications (PTMs) with specific functional states of chromatin is well established.6,7 Modification specific antibodies have been derived to study histone PTMs including acetylation, methylation, phosphorylation, or ubiquitination of specific residues. These antibodies can be very sensitive and specific,8 but the quantitative extent to which a given modification applies is difficult to determine. Also, while chromatin immunoprecipitation and reprecipitation techniques can be used to detect different modifications in the vicinity of each other,9,10 these techniques are laborious and cannot readily detect multiple different modifications present on the same molecule, a potentially important functional signature. For example the pattern of multiple modifications in individual histones, and their association in nucleosomes, appears to be central to epigenetic regulation of DNA function in health and disease,2,4,11–14 providing a combinatorial “histone code” specifying chromatin function.7,15 A sensitive, specific, and quantitative method to systematically characterize histone PTMs (4) (5) (6) (7)
(8)
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(10) (11)
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McNairn, A. J.; Gilbert, D. M. BioEssays 2003, 25, 647–56. Sproul, D.; Gilbert, N.; Bickmore, W. A. Nat. Rev. Genet. 2005, 6, 775–81. Jenuwein, T.; Allis, C. D. Science 2001, 293, 1074–80. Rea, S.; Eisenhaber, F.; O’Carroll, D.; Strahl, B. D.; Sun, Z. W.; Schmid, M.; Opravil, S.; Mechtler, K.; Ponting, C. P.; Allis, C. D.; Jenuwein, T. Nature 2000, 406, 593–9. Taverna, S. D.; Ueberheide, B. M.; Liu, Y.; Tackett, A. J.; Diaz, R. L.; Shabanowitz, J.; Chait, B. T.; Hunt, D. F.; Allis, C. D. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2086–91. Bernstein, B. E.; Mikkelsen, T. S.; Xie, X.; Kamal, M.; Huebert, D. J.; Cuff, J.; Fry, B.; Meissner, A.; Wernig, M.; Plath, K.; Jaenisch, R.; Wagschal, A.; Feil, R.; Schreiber, S. L.; Lander, E. S. Cell 2006, 125 (2), 315–26. Meissner, A.; Jaenisch, R. Nature 2006, 439 (7073), 212. Fraga, M. F.; Ballestar, E.; Paz, M. F.; Ropero, S.; Setien, F.; Ballestar, M. L.; Heine-Suner, D.; Cigudosa, J. C.; Urioste, M.; Benitez, J.; Boix-Chornet, M.; Sanchez-Aguilera, A.; Ling, C.; Carlsson, E.; Poulsen, P.; Vaag, A.; Stephan, Z.; Spector, T. D.; Wu, Y. Z.; Plass, C.; Esteller, M. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 10604–9. Fraga, M. F.; Esteller, M. Cell Cycle 2005, 4, 1377–81. Garcia, B. A.; Hake, S. B.; Diaz, R. L.; Kauer, M.; Morris, S. A.; Recht, J.; Shabanowitz, J.; Mishra, N.; Strahl, B. D.; Allis, C. D.; Hunt, D. F. J. Biol. Chem. 2007, 282 (10), 7641–55. Zhou, J.; Chau, C.; Deng, Z.; Stedman, W.; Lieberman, P. M. Cell Cycle 2005, 4, 889–92. Nightingale, K. P.; O’Neill, L. P.; Turner, B. M. Curr. Opin. Genet. Dev. 2006, 16, 125–36.
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Figure 3. 9.4 T compound FTICR mass spectrum of an isolated region of the 7+ charge state of histone H4, showing comparison with the simulated isotope distribution of the corresponding PTM states. Mass accuracy for each species is less than 2 ppm. Figure 1. Mean (n ) 4) MALDI-TOF mass spectra of acid extracted histones from untreated asynchronously growing wild type (dots) or p53 null (solid line) HCT116 colorectal cancer cells. Inset: mean (n ) 4) with error bars representing the standard error of the mean for MALDI-TOF mass spectra of histone H4, including post-translational modifications, from untreated asynchronously growing wild type (dots) or p53 null (solid line) HCT116 colorectal cancer cells. Note the apparent superposition of noncovalent sulfate adduct species on top of the underlying PTM distribution.
Figure 4. A 9.4 T compound FTICR mass spectrum of the 7+ charge state of histone H4 without TSA treatment, showing the observed post-translational modification distribution for the dominant N-terminally acetylated doubly methylated species. 9.4 T compound FTICR mass spectrum of the 7+ charge state of histone H4 following 3 h of 500 nM TSA treatment, showing the observed post-translational modification distribution for the dominant N-terminally acetylated, quadruply lysine acetylated, and doubly methylated species. Figure 2. 9.4 T broadband FTICR mass spectrum of the charge state distributions of the core histones present in a typical acid extraction from HCT116 cells, illustrating the reduced charge states and high background associated with contamination by low molecular weight organic compounds. Inset: 9.4 T broadband FTICR mass spectrum of the charge state distributions of the core histones present in a typical LC fraction of an acid extract from HCT116 cells, illustrating the broader charge states distribution and reduced background associated with elimination of low molecular weight organic compounds.
and especially their combinations in the individual histone molecule would therefore be of value. The various covalent histone modifications are catalyzed by families of enzymes which add or remove the relevant modification in a highly regulated process. In the case of histone acetylation, acetyl groups are added by histone acetyl transferases and removed by histone deacetylases. Trichostatin A (TSA) inhibits histone deacetylases at concentrations which are otherwise well tolerated by living cells, making it a useful drug for manipulating histone acetylation status experimentally in cell culture. Recent advances in mass spectrometry (MS) provide direct methods for detecting histone PTMs by characteristic mass shifts 4148
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in the intact proteins or their constituent peptides, the latter generated by physical fragmentation or by enzymatic digestion.8,16–20 To date, these methods have tended to use preseparation of acid extracted histone mixtures, or their digested peptide products, using liquid chromatography.21,22 Prior digestion yields fragments of smaller mass amenable to mass analysis on a wider variety of mass spectrometers but may dissociate coexistent PTMs present in different parts of the intact protein and frequently also results in incomplete coverage of the primary protein sequence when (16) Freitas, M. A.; Sklenar, A. R.; Parthun, M. R. J. Cell. Biochem. 2004, 92, 691–700. (17) Garcia, B. A.; Shabanowitz, J.; Hunt, D. F. Curr. Opin. Chem. Biol. 2007, 11, 66–73. (18) Hyland, E. M.; Cosgrove, M. S.; Molina, H.; Wang, D.; Pandey, A.; Cottee, R. J.; Boeke, J. D. Mol. Cell. Biol. 2005, 25, 10060–70. (19) Naldi, M.; Andrisano, V.; Fiori, J.; Calonghi, N.; Pagnotta, E.; Parolin, C.; Pieraccini, G.; Masotti, L. J. Chromatogr., A 2006, 1129, 73–81. (20) Smith, C. M.; Gafken, P. R.; Zhang, Z.; Gottschling, D. E.; Smith, J. B.; Smith, D. L. Anal. Biochem. 2003, 316, 23–33. (21) Bonenfant, D.; Coulot, M.; Towbin, H.; Schindler, P.; van Oostrum, J. Mol. Cell. Proteomics 2006, 5, 541–2. (22) Zhang, K.; Yau, P. M.; Chandrasekhar, B.; New, R.; Kondrat, R.; Imai, B. S.; Bradbury, M. E. Proteomics 2004, 4, 1–10.
Figure 5. Time course of evolution of 9.4 T FTICR mass spectrum of the 7+ charge state of histone H4 during treatment of HCT116 cells with TSA. Dose response of 9.4 T FTICR mass spectrum of the 7+ charge state of histone H4 following 3 h of treatment of HCT116 cells with TSA. Time course of evolution of 9.4 T FTICR mass spectrum of the 7+ charge state of histone H4 after removal of the drug following 3 h treatment of HCT116 cells with 500 nM TSA.
Figure 6. Mean and standard error of mean (n ) 3) for integrated abundance of each H4 ion species (every species is N-terminally acetylated) for p53 null (solid bars) and wild type (open bars) HCT116 without TSA. Mean and standard error of mean (n ) 3) for integrated abundance of each H4 ion species (every species is N-terminally acetylated) for p53 null (solid bars) and wild type (open bars) HCT116 after 3 h of 500 nM TSA.
particular peptide species are detected poorly in the mass spectrometer. Chromatographic preseparation simplifies the observed mass spectra improving the specificity of assignment of ion species22 but may degrade the quantitative reproducibility of the technique. In these cases, chemical derivitization has often been used to improve quantitation.23,24 Mass analysis of intact proteins using high mass resolution MS, for example, Fourier
transform ion cyclotron resonance (FTICR), avoids the problems of prior digestion and identifies coexistent PTMs in the individual protein molecule by characteristic mass shifts.25–29 In this paper, we further develop these FTICR MS techniques in order to determine the sequence and relative extent to which dominant PTMs occur in individual histone H4 molecules under varying experimental conditions. By eliminating preseparation, we obtain a global view of all four core histones and their PTMs. Focusing on histone H4, we demonstrate a technique that offers rapid analysis and relative quantitatation for the distribution of histone H4 PTMs in untreated cultured cancer cells and following histone deacetylase (HDAC) inhibitor (trichostatin A) treatment. To address the specificity of PTM assignments, we develop a novel preseparation process using a monolithic PF-DVB column (Dionex, Sunnyvale, CA) interfaced to a 12 T Apex-Qe Fourier transform ion cyclotron resonance mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with electron capture dissociation (ECD) yielding good sensitivity and Bonaldi, T.; Imhof, A.; Regula, J. T. Proteomics 2004, 4, 1382–96. Smith, C. M. Methods 2005, 36, 395–403. Galasinski, S. C.; Resing, K. A.; Ahn, N. G. Methods 2003, 31, 3–11. Thomas, C. E.; Kelleher, N. L.; Mizzen, C. A. J. Proteome Res. 2006, 5, 240–7. (27) Boyne, M. T.; Pesavento, J. J.; Mizzen, C. A.; Kelleher, N. L. J. Proteome Res. 2006, 5, 248–53. (28) Pesavento, J. J.; Kim, Y. B.; Taylor, G. K.; Kelleher, N. L. J. Am. Chem. Soc. 2004, 126, 3386–7. (29) Medzihradszky, K. F.; Zhang, X.; Chalkley, R. J.; Guan, S.; McFarland, M. A.; Chalmers, M. J.; Marshall, A. G.; Diaz, R. L.; Allis, C. D.; Burlingame, A. L. Mol. Cell. Proteomics 2004, 3 (9), 872–86. (23) (24) (25) (26)
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Figure 7. nLC fraction (3 µL) 12 T FTICR mass spectra of the 13+ charge state of histone H4 (treated with 10 nM TSA for 3 h) illustrating the isolated region for ECD fragmentation. ECD fragmentation spectrum of the quadrupolar isolated N terminally acetylated and doubly methylated species of histone H4. N-terminal containing ions designated “c” and C-terminal containing ions designated “z”. Fragmentation pattern as identified by Prosight PTM (mass accuracy
