Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Unabridged Analysis of Human Histone H3 by Differential Top-Down Mass Spectrometry Reveals Hypermethylated Proteoforms from MMSET/NSD2 Overexpression

2015; Elsevier BV; Volume: 15; Issue: 3 Linguagem: Inglês

10.1074/mcp.m115.053819

ISSN

1535-9484

Autores

Yupeng Zheng, Luca Fornelli, Philip D. Compton, S. Sharma, Jesse D. Canterbury, Christopher Mullen, Vlad Zabrouskov, Ryan T. Fellers, Paul M. Thomas, Jonathan D. Licht, Michael W. Senko, Neil L. Kelleher,

Tópico(s)

RNA modifications and cancer

Resumo

Histones, and their modifications, are critical components of cellular programming and epigenetic inheritance. Recently, cancer genome sequencing has uncovered driver mutations in chromatin modifying enzymes spurring high interest how such mutations change histone modification patterns. Here, we applied Top-Down mass spectrometry for the characterization of combinatorial modifications (i.e. methylation and acetylation) on full length histone H3 from human cell lines derived from multiple myeloma patients with overexpression of the histone methyltransferase MMSET as the result of a t(4;14) chromosomal translocation. Using the latest in Orbitrap-based technology for clean isolation of isobaric proteoforms containing up to 10 methylations and/or up to two acetylations, we provide extensive characterization of histone H3.1 and H3.3 proteoforms. Differential analysis of modifications by electron-based dissociation recapitulated antagonistic crosstalk between K27 and K36 methylation in H3.1, validating that full-length histone H3 (15 kDa) can be analyzed with site-specific assignments for multiple modifications. It also revealed K36 methylation in H3.3 was affected less by the overexpression of MMSET because of its higher methylation levels in control cells. The co-occurrence of acetylation with a minimum of three methyl groups in H3K9 and H3K27 suggested a hierarchy in the addition of certain modifications. Comparative analysis showed that high levels of MMSET in the myeloma-like cells drove the formation of hypermethyled proteoforms containing H3K36me2 co-existent with the repressive marks H3K9me2/3 and H3K27me2/3. Unique histone proteoforms with such "trivalent hypermethylation" (K9me2/3-K27me2/3-K36me2) were not discovered when H3.1 peptides were analyzed by Bottom-Up. Such disease-correlated proteoforms could link tightly to aberrant transcription programs driving cellular proliferation, and their precise description demonstrates that Top-Down mass spectrometry can now decode crosstalk involving up to three modified sites. Histones, and their modifications, are critical components of cellular programming and epigenetic inheritance. Recently, cancer genome sequencing has uncovered driver mutations in chromatin modifying enzymes spurring high interest how such mutations change histone modification patterns. Here, we applied Top-Down mass spectrometry for the characterization of combinatorial modifications (i.e. methylation and acetylation) on full length histone H3 from human cell lines derived from multiple myeloma patients with overexpression of the histone methyltransferase MMSET as the result of a t(4;14) chromosomal translocation. Using the latest in Orbitrap-based technology for clean isolation of isobaric proteoforms containing up to 10 methylations and/or up to two acetylations, we provide extensive characterization of histone H3.1 and H3.3 proteoforms. Differential analysis of modifications by electron-based dissociation recapitulated antagonistic crosstalk between K27 and K36 methylation in H3.1, validating that full-length histone H3 (15 kDa) can be analyzed with site-specific assignments for multiple modifications. It also revealed K36 methylation in H3.3 was affected less by the overexpression of MMSET because of its higher methylation levels in control cells. The co-occurrence of acetylation with a minimum of three methyl groups in H3K9 and H3K27 suggested a hierarchy in the addition of certain modifications. Comparative analysis showed that high levels of MMSET in the myeloma-like cells drove the formation of hypermethyled proteoforms containing H3K36me2 co-existent with the repressive marks H3K9me2/3 and H3K27me2/3. Unique histone proteoforms with such "trivalent hypermethylation" (K9me2/3-K27me2/3-K36me2) were not discovered when H3.1 peptides were analyzed by Bottom-Up. Such disease-correlated proteoforms could link tightly to aberrant transcription programs driving cellular proliferation, and their precise description demonstrates that Top-Down mass spectrometry can now decode crosstalk involving up to three modified sites. The field of epigenetics has seen an explosion of research in the past decade as scientists from different fields discovered its critical roles in many aspects related to human health, ranging from stem cell pluripotency to aging (1.Ladewig J. Koch P. Brustle O. Leveling Waddington: the emergence of direct programming and the loss of cell fate hierarchies.Nat. Rev. Mol. Cell Biol. 2013; 14: 225-236Crossref Scopus (174) Google Scholar, 2.López-Otín C. Blasco M.A. Partridge L. Serrano M. Kroemer G. The hallmarks of aging.Cell. 2013; 153: 1194-1217Abstract Full Text Full Text PDF PubMed Scopus (7742) Google Scholar, 3.Greer E.L. Maures T.J. Ucar D. Hauswirth A.G. Mancini E. Lim J.P. Benayoun B.A. Shi Y. Brunet A. Transgenerational epigenetic inheritance of longevity in Caenorhabditis elegans.Nature. 2011; 479: 365-371Crossref PubMed Scopus (451) Google Scholar), from cancer to microbial infection (4.Béguelin W. Popovic R. Teater M. Jiang Y. Bunting K.L. Rosen M. Shen H. Yang S.N. Wang L. Ezponda T. Martinez-Garcia E. Zhang H. Zheng Y. Verma S.K. McCabe M.T. Ott H.M. Van Aller G.S. Kruger R.G. Liu Y. McHugh C.F. Scott D.W. Chung Y.R. Kelleher N. Shaknovich R. Creasy C.L. Gascoyne R.D. Wong K.K. Cerchietti L. Levine R.L. Abdel-Wahab O. Licht J.D. Elemento O. Melnick A.M. EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation.Cancer Cell. 2013; 23: 677-692Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar, 5.Morin R.D. Johnson N.A. Severson T.M. Mungall A.J. An J. Goya R. Paul J.E. Boyle M. Woolcock B.W. Kuchenbauer F. Yap D. Humphries R.K. Griffith O.L. Shah S. Zhu H. Kimbara M. Shashkin P. Charlot J.F. Tcherpakov M. Corbett R. Tam A. Varhol R. Smailus D. Moksa M. Zhao Y. Delaney A. Qian H. Birol I. Schein J. Moore R. Holt R. Horsman D.E. Connors J.M. Jones S. Aparicio S. Hirst M. Gascoyne R.D. Marra M.A. Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin.Nat. Genet. 2010; 42: 181-185Crossref PubMed Scopus (1311) Google Scholar, 6.Sneeringer C.J. Scott M.P. Kuntz K.W. Knutson S.K. Pollock R.M. Richon V.M. Copeland R.A. Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 20980-20985Crossref PubMed Scopus (525) Google Scholar, 7.Schwartzentruber J. Korshunov A. Liu X.-Y. Jones D.T.W. Pfaff E. Jacob K. Sturm D. Fontebasso A.M. Quang D.-A. K. Tonjes M. Hovestadt V. Albrecht S. Kool M. Nantel A. Konermann C. Lindroth A. Jäger N. Rausch T. Ryzhova M. Korbel J.O. Hielscher T. Hauser P. Garami M. Klekner A. Bognar L. Ebinger M. Schuhmann M.U. Scheurlen W. Pekrun A. Fruhwald M.C. Roggendorf W. Kramm C. Durken M. Atkinson J. Lepage P. Montpetit A. Zakrzewska M. Zakrzewski K. Liberski P.P. Dong Z. Siegel P. Kulozik A.E. Zapatka M. Guha A. Malkin D. Felsberg J. Reifenberger G. von Deimling A. Ichimura K. Collins V.P. Witt H. Milde T. Witt O. Zhang C. Castelo-Branco P. Lichter P. Faury D. Tabori U. Plass C. Majewski J. Pfister S.M. Jabado N. Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma.Nature. 2012; 482: 226-231Crossref PubMed Scopus (1707) Google Scholar, 8.Jaffe J.D. Wang Y. Chan H.M. Zhang J. Huether R. Kryukov G.V. Bhang H.E. Taylor J.E. Hu M. Englund N.P. Yan F. Wang Z. Robert McDonald 3rd, E. Wei L. Ma J. Easton J. Yu Z. deBeaumount R. Gibaja V. Venkatesan K. Schlegel R. Sellers W.R. Keen N. Liu J. Caponigro G. Barretina J. Cooke V.G. Mullighan C. Carr S.A. Downing J.R. Garraway L.A. Stegmeier F. Global chromatin profiling reveals NSD2 mutations in pediatric acute lymphoblastic leukemia.Nat. Genet. 2013; 45: 1386-1391Crossref PubMed Scopus (185) Google Scholar), from memory processing to drug addiction (9.Gräff J. Tsai L.H. Histone acetylation: molecular mnemonics on the chromatin.Nat. Rev. Neurosci. 2013; 14: 97-111Crossref PubMed Scopus (426) Google Scholar, 10.Schmidt H.D. McGinty J.F. West A.E. Sadri-Vakili G. Epigenetics and psychostimulant addiction.Perspectives Med. 2013; 3: a012047Google Scholar). Histone modifications, including methylation (me), acetylation (ac), monoubiquitylation (ub1), etc., are related to the study of epigenetics (11.Badeaux A.I. Shi Y. Emerging roles for chromatin as a signal integration and storage platform.Nat. Rev. Mol. Cell Biol. 2013; 14: 211-224Crossref Scopus (214) Google Scholar, 12.Berger S.L. Kouzarides T. Shiekhattar R. Shilatifard A. An operational definition of epigenetics.Genes Develop. 2009; 23: 781-783Crossref PubMed Scopus (1239) Google Scholar). These modifications, as well as their different states in case of methylation (i.e. mono-, di-, and trimethylation) and positions on the histone, play important and distinct roles in almost every activity operative on the chromatin template. The significance of these modifications are further underscored by the unexpected identification of many driver mutations underlying cancer biology within histone modifying enzymes (13.Zaidi S. Choi M. Wakimoto H. Ma L. Jiang J. Overton J.D. Romano-Adesman A. Bjornson R.D. Breitbart R.E. Brown K.K. Carriero N.J. Cheung Y.H. Deanfield J. DePalma S. Fakhro K.A. Glessner J. Hakonarson H. Italia M.J. Kaltman J.R. Kaski J. Kim R. Kline J.K. Lee T. Leipzig J. Lopez A. Mane S.M. Mitchell L.E. Newburger J.W. Parfenov M. Pe'er I. Porter G. Roberts A.E. Sachidanandam R. Sanders S.J. Seiden H.S. State M.W. Subramanian S. Tikhonova I.R. Wang W. Warburton D. White P.S. Williams I.A. Zhao H. Seidman J.G. Brueckner M. Chung W.K. Gelb B.D. Goldmuntz E. Seidman C.E. Lifton R.P. De novo mutations in histone-modifying genes in congenital heart disease.Nature. 2013; 498: 220-223Crossref PubMed Scopus (630) Google Scholar, 14.Suvà M.L. Riggi N. Bernstein B.E. Epigenetic reprogramming in cancer.Science. 2013; 339: 1567-1570Crossref PubMed Scopus (529) Google Scholar) and somatic mutations in histone H3.3 (7.Schwartzentruber J. Korshunov A. Liu X.-Y. Jones D.T.W. Pfaff E. Jacob K. Sturm D. Fontebasso A.M. Quang D.-A. K. Tonjes M. Hovestadt V. Albrecht S. Kool M. Nantel A. Konermann C. Lindroth A. Jäger N. Rausch T. Ryzhova M. Korbel J.O. Hielscher T. Hauser P. Garami M. Klekner A. Bognar L. Ebinger M. Schuhmann M.U. Scheurlen W. Pekrun A. Fruhwald M.C. Roggendorf W. Kramm C. Durken M. Atkinson J. Lepage P. Montpetit A. Zakrzewska M. Zakrzewski K. Liberski P.P. Dong Z. Siegel P. Kulozik A.E. Zapatka M. Guha A. Malkin D. Felsberg J. Reifenberger G. von Deimling A. Ichimura K. Collins V.P. Witt H. Milde T. Witt O. Zhang C. Castelo-Branco P. Lichter P. Faury D. Tabori U. Plass C. Majewski J. Pfister S.M. Jabado N. Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma.Nature. 2012; 482: 226-231Crossref PubMed Scopus (1707) Google Scholar, 15.Behjati S. Tarpey P.S. Presneau N. Scheipl S. Pillay N. Van Loo P. Wedge D.C. Cooke S.L. Gundem G. Davies H. Nik-Zainal S. Martin S. McLaren S. Goodie V. Robinson B. Butler A. Teague J.W. Halai D. Khatri B. Myklebost O. Baumhoer D. Jundt G. Hamoudi R. Tirabosco R. Amary M.F. Futreal P.A. Stratton M.R. Campbell P.J. Flanagan A.M. Distinct H3F3A and H3F3B driver mutations define chondroblastoma and giant cell tumor of bone.Nat. Genet. 2013; 45: 1479-1482Crossref PubMed Scopus (525) Google Scholar, 16.Herz H.M. Morgan M. Gao X. Jackson J. Rickels R. Swanson S.K. Florens L. Washburn M.P. Eissenberg J.C. Shilatifard A. Histone H3 lysine-to-methionine mutants as a paradigm to study chromatin signaling.Science. 2014; 345: 1065-1070Crossref PubMed Scopus (130) Google Scholar). Widely used antibody-based measurements of histone modifications face two analytical challenges: (1) similar chemical structure of modification (e.g. three distinct methylation states of mono-, di-, and tri-methylation) and closely related flanking sequence can lead to cross-reactivity (17.Peach S.E. Rudomin E.L. Udeshi N.D. Carr S.A. Jaffe J.D. Quantitative assessment of chromatin immunoprecipitation grade antibodies directed against histone modifications reveals patterns of co-occurring marks on histone protein molecules.Mol. Cell. Proteomics. 2012; 11: 128-137Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar); (2) close proximity of many modifications can have unexpected effects in antibody recognition (18.Fuchs S.M. Krajewski K. Baker R.W. Miller V.L. Strahl B.D. Influence of combinatorial histone modifications on antibody and effector protein recognition.Curr. Biol. 2011; 21: 53-58Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). For example, H4K20me2 antibody can lose its recognition when acetylation is present in the neighboring H4K16 (19.Hsiao K.Y. Mizzen C.A. Histone H4 deacetylation facilitates 53BP1 DNA damage signaling and double-strand break repair.J. Mol. Cell Biol. 2013; 5: 157-165Crossref PubMed Scopus (124) Google Scholar). Therefore, analyzing histone modifications by MS can provide a highly valuable orthogonal measurement. There are two general modes of interrogation by MS: Bottom-Up analysis of tryptic peptides, and Top-Down or Middle-Down measurement of full-length histones or large tail peptides, respectively (20.Yuan Z.F. Arnaudo A.M. Garcia B.A. Mass spectrometric analysis of histone proteoforms.Annu. Rev. Anal. Chem. 2014; 7: 113-128Crossref PubMed Scopus (46) Google Scholar). The value of mass spectrometric analysis of histone modifications in the field of cancer epigenetics was further demonstrated by the recent identification of a recurrent point mutation of E1099K in MMSET in lymphoid malignancies (8.Jaffe J.D. Wang Y. Chan H.M. Zhang J. Huether R. Kryukov G.V. Bhang H.E. Taylor J.E. Hu M. Englund N.P. Yan F. Wang Z. Robert McDonald 3rd, E. Wei L. Ma J. Easton J. Yu Z. deBeaumount R. Gibaja V. Venkatesan K. Schlegel R. Sellers W.R. Keen N. Liu J. Caponigro G. Barretina J. Cooke V.G. Mullighan C. Carr S.A. Downing J.R. Garraway L.A. Stegmeier F. Global chromatin profiling reveals NSD2 mutations in pediatric acute lymphoblastic leukemia.Nat. Genet. 2013; 45: 1386-1391Crossref PubMed Scopus (185) Google Scholar, 21.Oyer J.A. Huang X. Zheng Y. Shim J. Ezponda T. Carpenter Z. Allegretta M. Okot-Kotber C.I. Patel J.P. Melnick A. Levine R.L. Ferrando A. Mackerell Jr., A.D. Kelleher N.L. Licht J.D. Popovic R. Point mutation E1099K in MMSET/NSD2 enhances its methyltranferase activity and leads to altered global chromatin methylation in lymphoid malignancies.Leukemia. 2014; 28: 198-201Crossref PubMed Scopus (94) Google Scholar). The "histone code" hypothesis posits that the combinatorial nature of histone modifications can serve as the binding platform to elicit specific cellular processes (22.Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7632) Google Scholar). Recently, the combination of modifications has been used to define chromatin states, which are generated from meta-analysis of multiple ChIP-Seq data sets and found to be highly dynamic among different cell lines (23.Ernst J. Kheradpour P. Mikkelsen T.S. Shoresh N. Ward L.D. Epstein C.B. Zhang X. Wang L. Issner R. Coyne M. Ku M. Durham T. Kellis M. Bernstein B.E. Mapping and analysis of chromatin state dynamics in nine human cell types.Nature. 2011; 473: 43-49Crossref PubMed Scopus (2063) Google Scholar). However, this type of antibody-based technique relies on the associated DNA sequence to infer PTM co-occurrence from an average of histone modifications in the same locus but not necessarily on the same molecule. MMSET (also known as NSD2 or WHSC1) is one of the eight known histone methyltransferases targeting H3K36 (24.Wagner E.J. Carpenter P.B. Understanding the language of Lys36 methylation at histone H3.Nat. Rev. Mol. Cell Biol. 2012; 13: 115-126Crossref PubMed Scopus (632) Google Scholar) with specificity toward dimethylation (25.Li Y. Trojer P. Xu C.F. Cheung P. Kuo A. Drury 3rd, W.J. Qiao Q. Neubert T.A. Xu R.M. Gozani O. Reinberg D. The target of the NSD family of histone lysine methyltransferases depends on the nature of the substrate.J. Biol. Chem. 2009; 284: 34283-34295Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Overexpression of MMSET has been documented in ∼20% of multiple myeloma cases as the result of chromosomal translocation t(4;14) (26.Keats J.J. Reiman T. Belch A.R. Pilarski L.M. Ten years and counting: so what do we know about t(4;14)(p16;q32) multiple myeloma.Leuk. Lymphoma. 2006; 47: 2289-2300Crossref PubMed Scopus (75) Google Scholar), which places the MMSET gene under the strong immunoglobulin enhancers (27.Stec I. Wright T.J. van Ommen G.J. de Boer P.A. van Haeringen A. Moorman A.F. Altherr M.R. den Dunnen J.T. WHSC1, a 90 kb SET domain-containing gene, expressed in early development and homologous to a Drosophila dysmorphy gene maps in the Wolf-Hirschhorn syndrome critical region and is fused to IgH in t(4;14) multiple myeloma.Human Mol. Genet. 1998; 7: 1071-1082Crossref PubMed Scopus (251) Google Scholar). A pair of cell lines, TKO and NTKO, were engineered from a t(4;14)+ multiple myeloma patient-derived cell line, KMS11. In the targeted knockout (TKO) 1The abbreviations used are:TKOtargeted knockoutHILIChydrophilic interaction chromatographyLCliquid chromatographyMSmass spectrometryETDelectron transfer dissociationEThcDelectron transfer dissociation – higher-energy collision dissociation. cell line, the translocated copy of MMSET was knocked out by homologous recombination, which leads to close to normal expression level of MMSET. In the nontargeted knockout (NTKO) cell line, the non-translocated gene was knocked out and the expression level of MMSET remains high (28.Lauring J. Abukhdeir A.M. Konishi H. Garay J.P. Gustin J.P. Wang Q. Arceci R.J. Matsui W. Park B.H. The multiple myeloma associated MMSET gene contributes to cellular adhesion, clonogenic growth, and tumorigenicity.Blood. 2008; 111: 856-864Crossref PubMed Scopus (129) Google Scholar). Our quantitative Bottom-Up MS assay using selective reaction monitoring revealed how overexpression of a HMT targeting H3K36 led to the global changes in both H3K27 and H3K36 methylation (29.Zheng Y. Sweet S.M. Popovic R. Martinez-Garcia E. Tipton J.D. Thomas P.M. Licht J.D. Kelleher N.L. Total kinetic analysis reveals how combinatorial methylation patterns are established on lysines 27 and 36 of histone H3.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 13549-13554Crossref PubMed Scopus (93) Google Scholar, 30.Popovic R. Martinez-Garcia E. Giannopoulou E.G. Zhang Q. Ezponda T. Shah M.Y. Zheng Y. Will C.M. Small E.C. Hua Y. Bulic M. Jiang Y. Carrara M. Calogero R.A. Kath W.L. Kelleher N.L. Wang J.P. Elemento O. Licht J.D. Histone methyltransferase MMSET/NSD2 alters EZH2 binding and reprograms the myeloma epigenome through global and focal changes in H3K36 and H3K27 methylation.PLoS Genet. 2014; 10: e1004566Crossref PubMed Scopus (148) Google Scholar, 31.Kuo A.J. Cheung P. Chen K. Zee B.M. Kioi M. Lauring J. Xi Y. Park B.H. Shi X. Garcia B.A. Li W. Gozani O. NSD2 links dimethylation of histone H3 at lysine 36 to oncogenic programming.Mol. Cell. 2011; 44: 609-620Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Here, our goal was to make differential Top-Down measurement of two histone H3 variants, whose synthesis is (H3.1) and is not (H3.3) dependent on replication during S phase (32.Filipescu D. Muller S. Almouzni G. Histone H3 variants and their chaperones during development and disease: contributing to epigenetic control.Ann. Rev. Cell Develop. Biol. 2014; 30: 615-646Crossref PubMed Scopus (77) Google Scholar). targeted knockout hydrophilic interaction chromatography liquid chromatography mass spectrometry electron transfer dissociation electron transfer dissociation – higher-energy collision dissociation. To directly catalogue modifications co-occurring on the same histone (i.e. combinatorial modifications), we have reported Top-Down MS analysis of all histones (33.Pesavento J.J. Mizzen C.A. Kelleher N.L. Quantitative analysis of modified proteins and their positional isomers by tandem mass spectrometry: human histone H4.Anal. Chem. 2006; 78: 4271-4280Crossref PubMed Scopus (201) Google Scholar, 34.Boyne 2nd, M.T. Pesavento J.J. Mizzen C.A. Kelleher N.L. Precise characterization of human histones in the H2A gene family by top down mass spectrometry.J. Proteome Res. 2006; 5: 248-253Crossref PubMed Scopus (142) Google Scholar, 35.Siuti N. Roth M.J. Mizzen C.A. Kelleher N.L. Pesavento J.J. Gene-specific characterization of human histone H2B by electron capture dissociation.J. Proteome Res. 2006; 5: 233-239Crossref PubMed Scopus (94) Google Scholar, 36.Thomas C.E. Kelleher N.L. Mizzen C.A. Mass spectrometric characterization of human histone H3: a bird's eye view.J. Proteome Res. 2006; 5: 240-247Crossref PubMed Scopus (169) Google Scholar, 37.Zheng Y. John S. Pesavento J.J. Schultz-Norton J.R. Schiltz R.L. Baek S. Nardulli A.M. Hager G.L. Kelleher N.L. Mizzen C.A. Histone H1 phosphorylation is associated with transcription by RNA polymerases I and II.J. Cell Biol. 2010; 189: 407-415Crossref PubMed Scopus (81) Google Scholar) and Middle-Down MS for 1–50 N-terminal piece of histone H3 (5.3 kDa) (38.Garcia B.A. Pesavento J.J. Mizzen C.A. Kelleher N.L. Pervasive combinatorial modification of histone H3 in human cells.Nat. Methods. 2007; 4: 487-489Crossref PubMed Scopus (195) Google Scholar). Great efforts from many laboratories also continue to improve the utility of Top-Down and Middle-Down MS for histone analysis (39.Phanstiel D. Brumbaugh J. Berggren W.T. Conard K. Feng X. Levenstein M.E. McAlister G.C. Thomson J.A. Coon J.J. Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 4093-4098Crossref PubMed Scopus (142) Google Scholar, 40.Tian Z. Tolic N. Zhao R. Moore R.J. Hengel S.M. Robinson E.W. Stenoien D.L. Wu S. Smith R.D. Pasa-Tolic L. Enhanced top-down characterization of histone post-translational modifications.Genome Biol. 2012; 13: R86Crossref PubMed Google Scholar, 41.Moradian A. Kalli A. Sweredoski M.J. Hess S. The top-down, middle-down, and bottom-up mass spectrometry approaches for characterization of histone variants and their post-translational modifications.Proteomics. 2014; 14: 489-497Crossref PubMed Scopus (113) Google Scholar, 42.Sidoli S. Lin S. Karch K.R. Garcia B.A. Bottom-up and middle-down proteomics have comparable accuracies in defining histone post-translational modification relative abundance and stoichiometry.Anal. Chem. 2015; 87: 3129-3133Crossref PubMed Scopus (44) Google Scholar, 43.Harshman S.W. Hoover M.E. Huang C. Branson O.E. Chaney S.B. Cheney C.M. Rosol T.J. Shapiro C.L. Wysocki V.H. Huebner K. Freitas M.A. Histone H1 phosphorylation in breast cancer.J.Proteome Res. 2014; 13: 2453-2467Crossref PubMed Scopus (31) Google Scholar, 44.Young N.L. DiMaggio P.A. Plazas-Mayorca M.D. Baliban R.C. Floudas C.A. Garcia B.A. High throughput characterization of combinatorial histone codes.Mol. Cell. Proteomics. 2009; 8: 2266-2284Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). However, it is still very challenging to apply Top-Down approach for the routine analysis of histone proteoforms (45.Smith L.M. Kelleher N.L. Proteoform: a single term describing protein complexity.Nat. Methods. 2013; 10: 186-187Crossref PubMed Scopus (884) Google Scholar). This was demonstrated in the first pilot project from the Consortium for Top-Down Proteomics to assess intra-laboratory variation in the characterization of histone H4 (46.Dang X. Scotcher J. Wu S. Chu R.K. Tolic N. Ntai I. Thomas P.M. Fellers R.T. Early B.P. Zheng Y. Durbin K.R. Leduc R.D. Wolff J.J. Thompson C.J. Pan J. Han J. Shaw J.B. Salisbury J.P. Easterling M. Borchers C.H. Brodbelt J.S. Agar J.N. Pasa-Tolic L. Kelleher N.L. Young N.L. The first pilot project of the consortium for top-down proteomics: a status report.Proteomics. 2014; 14: 1130-1140Crossref PubMed Scopus (77) Google Scholar). One of the key limitations identified in that study was a need for continued improvement in high-resolution isolation and high-efficiency fragmentation. These two critical aspects for high quality proteoform characterization align with the development of a new Orbitrap-based tribrid mass spectrometer, whose architecture includes a segmented quadrupole for narrow precursor isolation with high transmission efficiency, improved vacuum conditions, and the optimization of multiple ion dissociation techniques including electron transfer dissociation (ETD) (47.Syka J.E. Coon J.J. Schroeder M.J. Shabanowitz J. Hunt D.F. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9528-9533Crossref PubMed Scopus (1999) Google Scholar), higher-energy collisional dissociation (HCD) (48.Olsen J.V. Macek B. Lange O. Makarov A. Horning S. Mann M. Higher-energy C-trap dissociation for peptide modification analysis.Nat. Methods. 2007; 4: 709-712Crossref PubMed Scopus (719) Google Scholar) and their combination (EThcD) (49.Frese C.K. Altelaar A.F. van den Toorn H. Nolting D. Griep-Raming J. Heck A.J. Mohammed S. Toward full peptide sequence coverage by dual fragmentation combining electron-transfer and higher-energy collision dissociation tandem mass spectrometry.Anal. Chem. 2012; 84: 9668-9673Crossref PubMed Scopus (216) Google Scholar, 50.Brunner A.M. Lossl P. Liu F. Huguet R. Mullen C. Yamashita M. Zabrouskov V. Makarov A. Altelaar A.F. Heck A.J. Benchmarking multiple fragmentation methods on an orbitrap fusion for top-down phospho-proteoform characterization.Anal. Chem. 2015; 87: 4152-4158Crossref PubMed Scopus (85) Google Scholar). Therefore, we aimed to develop proper workflow and informatic tools to enable Top-Down comparative interrogation of the most highly modified core histone, H3, upon cellular perturbation. KMS11 TKO and NTKO cells were cultured in RPMI media supplemented with 10% FBS (Sigma, St. Louis, MI). Harvested cells were flash frozen in liquid nitrogen and stored at −80 °C before sample preparation. Nuclei were isolated with NIB buffer (15 mm Tris, 60 mm KCl, 15 mm NaCl, 5 mm MgCl2, 1 mm CaCl2, 250 mm Sucrose, 0.5 mm 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 10 mm sodium butyrate, and 1 mm DTT, pH 7.5) containing 0.3% Nonidet P-40 and subsequently washed twice with NIB buffer without detergent. Isolated nuclei were recovered by centrifugation at 600 rcf. Crude histones were extracted from isolated nuclei with 0.4 N H2SO4 and recovered by precipitation with 20% (w/v, final) trichloroacetic acid (TCA). The precipitate was washed first with 0.1% HCl in acetone and twice with pure acetone. Crude histones were then resuspended in water and subjected to fractionation using reverse phase high pressure liquid chromatography (RP-HPLC) as described previously (51.Zheng Y. Tipton J.D. Thomas P.M. Kelleher N.L. Sweet S.M. Site-specific human histone H3 methylation stability: fast K4me3 turnover.Proteomics. 2014; 14: 2190-2199Crossref PubMed Scopus (17) Google Scholar). Briefly, histones were separated using a Jupiter C18 analytical column (Phenomenex, Torrance, CA), 15 cm × 4.6 mm, 5 μm diam., 300 Å pores, using a gradient of 30–57% B in 90 min (Buffer A: 5% ACN, 0.1% TFA; Buffer B: 90% ACN, 0.094% TFA) at a flow rate of 0.8 ml/min using an Agilent 1100 HPLC system (Agilent, Santa Clara, CA) monitored by UV absorbance at 214 nm. Fractionated histone H3.1 and H3.3 were collected and dried by speed-vacuum. Dried histone pellets were resuspended in 49.95:49.95:0.1 (v:v:v) water/acetonitrile/formic acid (LC-MS grade) at ∼1 μm final concentration, and were sprayed using a NanoFlex ion source (Thermo Fisher Scientific, San Jose, CA) equipped with a nanoelectrospray static probe and coated glass emitters, applying a 1.7–1.9 kV potential at the emitter. All mass spectrometry measurements were performed on an fETD-enabled (52.Earley L. Anderson L.C. Bai D.L. Mullen C. Syka J.E. English A.M. Dunyach J.J. Stafford Jr., G.C. Shabanowitz J. Hunt D.F. Compton P.D. Front-end electron transfer dissociation: a new ionization source.Anal. Chem. 2013; 85: 8385-8390Crossref PubMed Scopus (50) Google Scholar) Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) operating in Intact Protein Mode (N2 pressure at the ion routing multipole of 1 mTorr), using a resolving power of 60,000 (at m/z 200) and averaging five microscans for every scan, with transfer capillary temperature set at 275 °C, the RF of the source ion funnel operating at 20% and a source offset of 15 V to favor adduct removal. For each histone fraction, broadband MS1 spectra were recorded over a 500–2000 m/z window using an AGC target value of 2e5. MS1 spectra were used to define a list of histone proteoform peaks differing in mass for ∼14 Da, corresponding to the mass of one methylation. The list included m/z values corresponding to the most abundant isotopic distribution for each isobaric proteoform cluster. MS2 experiments were based on the isolation and subsequent fragmentation of each of these clusters for the 18+ precursor. Each proteoform cluster was quadrupole isolated using a 0.6 Th isolation window, and subjected sequentially first to high capacity ETD (ETD HD) performed with increasing duration, and then to EThcD, performed as previously described (50.Brunner A.M. Lossl P. Liu F. Huguet R. Mullen C. Yamashita M. Zabrouskov V. Makarov A. Altelaar A.F. Heck A.J. Benchmarking multiple fragmentation methods on an orbitrap fusion for top-down phospho-proteoform characterization.Anal. Chem. 2015; 87: 4152-4158Crossref PubMed Scop

Referência(s)