Collective mass spectrometry approaches reveal broad and combinatorial modification of high mobility group protein A1a

  • Nicolas L. Young
  • Mariana D. Plazas-Mayorca
  • Peter A. DiMaggio
  • Ian Z. Flaniken
  • Andrea J. Beltran
  • Neeli Mishra
  • Gary LeRoy
  • Christodoulos A. Floudas
  • Benjamin A. Garcia
Focus: Top-Down Mass Spectrometry

Abstract

Transcriptional states are formed and maintained by the interaction and post-translational modification (PTM) of several chromatin proteins, such as histones and high mobility group (HMG) proteins. Among these, HMGA1a, a small heterochromatin-associated nuclear protein has been shown to be post-translationally modified, and some of these PTMs have been linked to apoptosis and cancer. In cancerous cells, HMGA1a PTMs differ between metastatic and nonmetastatic cells, suggesting the existence of an HMGA1a PTM code analogous to the “histone code.” In this study, we expand on current knowledge by comprehensively characterizing PTMs on HMGA1a purified from human cells using both nanoflow liquid chromatography collision activated dissociation mediated Bottom Up and electron-transfer dissociation facilitated middle and Top Down mass spectrometry (MS). We find HMGA1a to be pervasively modified with many types of modifications such as methylation, acetylation, and phosphorylation, including finding novel sites. While Bottom Up MS identified lower level modification sites, Top and Middle Down MS were utilized to identify the most commonly occurring combinatorially modified forms. Remarkably, although we identify several individual modification sites through our Bottom Up and Middle Down MS analyses, we find relatively few combinatorially modified forms dominate the population through Top Down proteomics. The main combinatorial PTMs we find through the Top Down approach are N-terminal acetylation, Arg25 methylation along with phosphorylation of the three most C-terminal serine residues in primarily a diphosphorylated form. This report presents one of the most detailed analyses of HMGA1a to date and illustrates the strength of using a combined MS effort.

Supplementary material

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Supplementary material, approximately 33 KB.
13361_2011_210600960_MOESM2_ESM.pdf (39 kb)
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13361_2011_210600960_MOESM3_ESM.doc (311 kb)
Supplementary material, approximately 311 KB.

References

  1. 1.
    Bernstein, B. E.; Meissner, A.; Lander, E. S. The Mammalian Epigenome. Cell 2007, 128, 669–681.CrossRefGoogle Scholar
  2. 2.
    Cheung, P.; Allis, C. D.; Sassone-Corsi, P. Signaling to Chromatin through Histone Modifications. Cell 2000, 103, 263–271.CrossRefGoogle Scholar
  3. 3.
    Kouzarides, T. Chromatin Modifications and Their Function. Cell 2007, 128, 693–705.CrossRefGoogle Scholar
  4. 4.
    Lachner, M.; O’Carroll, D.; Rea, S.; Mechtler, K.; Jenuwein, T. Methylation of Histone H3 Lysine 9 Creates a Binding Site for Hp1 Proteins. Nature 2001, 410, 116–120.CrossRefGoogle Scholar
  5. 5.
    Bannister, A. J.; Zegerman, P.; Partridge, J. F.; Miska, E. A.; Thomas, J. O.; Allshire, R. C.; Kouzarides, T. Selective Recognition of Methylated Lysine 9 on Histone H3 by the Hp1 Chromo Domain. Nature 2001, 410, 120–124.CrossRefGoogle Scholar
  6. 6.
    Grosschedl, R.; Giese, K.; Pagel, J. HMG Domain Proteins: Architectural Elements in the Assembly of Nucleoprotein Structures. Trends Genet. 1994, 10, 94–100.CrossRefGoogle Scholar
  7. 7.
    Zhang, Q.; Wang, Y. High Mobility Group Proteins and Their Post-Translational Modifications. Biochim. Biophys. Acta 2008, 1784, 1159–1166.CrossRefGoogle Scholar
  8. 8.
    Chiappetta, G.; Bandiera, A.; Berlingieri, M. T.; Visconti, R.; Manfioletti, G.; Battista, S.; Martinez-Tello, F. J.; Santoro, M.; Giancotti, V.; Fusco, A. The Expression of the High Mobility Group Hmgi (Y) Proteins Correlates with the Malignant Phenotype of Human Thyroid Neoplasias. Oncogene 1995, 10, 1307–1314.Google Scholar
  9. 9.
    Giancotti, V.; Berlingieri, M. T.; DiFiore, P. P.; Fusco, A.; Vecchio, G.; Crane-Robinson, C. Changes in Nuclear Proteins on Transformation of Rat Epithelial Thyroid Cells by a Murine Sarcoma Retrovirus. Cancer Res. 1985, 45, 6051–6057.Google Scholar
  10. 10.
    Giancotti, V.; Buratti, E.; Perissin, L.; Zorzet, S.; Balmain, A.; Portella, G.; Fusco, A.; Goodwin, G. H. Analysis of the Hmg-I Nuclear Proteins in Mouse Neoplastic Cells Induced by Different Procedures. Exp. Cell. Res. 1989, 184, 538–545.CrossRefGoogle Scholar
  11. 11.
    Chiappetta, G.; Avantaggiato, V.; Visconti, R.; Fedele, M.; Battista, S.; Trapasso, F.; Merciai, B. M.; Fidanza, V.; Giancotti, V.; Santoro, M.; Simeone, A.; Fusco, A. High Level Expression of the Hmgi (Y) Gene During Embryonic Development. Oncogene 1996, 13, 2439–2446.Google Scholar
  12. 12.
    Zhou, X.; Benson, K. F.; Ashar, H. R.; Chada, K. Mutation Responsible for the Mouse Pygmy Phenotype in the Developmentally Regulated Factor Hmgi-C. Nature 1995, 376, 771–774.CrossRefGoogle Scholar
  13. 13.
    Amirand, C.; Viari, A.; Ballini, J. P.; Rezaei, H.; Beaujean, N.; Jullien, D.; Kas, E.; Debey, P. Three Distinct Sub-Nuclear Populations of Hmg-I Protein of Different Properties Revealed by Co-Localization Image Analysis. J. Cell Sci. 1998, 111(Pt. 23), 3551–3561.Google Scholar
  14. 14.
    Martelli, A. M.; Riccio, M.; Bareggi, R.; Manfioletti, G.; Tabellini, G.; Baldini, G.; Narducci, P.; Giancotti, V. Intranuclear Distribution of Hmgi/Y Proteins. An Immunocytochemical Study. J. Histochem. Cytochem. 1998, 46, 863–864.CrossRefGoogle Scholar
  15. 15.
    Reeves, R.; Wolffe, A. P. Substrate Structure Influences Binding of the Non-Histone Protein Hmg-I(Y) to Free Nucleosomal DNA. Biochemistry 1996, 35, 5063–5074.CrossRefGoogle Scholar
  16. 16.
    Reeves, R.; Nissen, M. S. Interaction of High Mobility Group-I (Y) Nonhistone Proteins with Nucleosome Core Particles. J. Biol. Chem. 1993, 268, 21137–21146.Google Scholar
  17. 17.
    Reeves, R.; Leonard, W. J.; Nissen, M. S. Binding of Hmg-I(Y) Imparts Architectural Specificity to a Positioned Nucleosome on the Promoter of the Human Interleukin-2 Receptor α Gene. Mol. Cell. Biol. 2000, 20, 4666–4679.CrossRefGoogle Scholar
  18. 18.
    Elton, T. S.; Reeves, R.; Purification and Postsynthetic Modifications of Friend Erythroleukemic Cell High Mobility Group Protein Hmg-I. Anal. Biochem. 1986, 157, 53–62.CrossRefGoogle Scholar
  19. 19.
    Lund, T.; Holtlund, J.; Laland, S. G. On the Phosphorylation of Low Molecular Mass Hmg (High Mobility Group) Proteins in Ehrlich Ascites Cells. FEBS Lett. 1985, 180, 275–279.CrossRefGoogle Scholar
  20. 20.
    Nissen, M. S.; Langan, T. A.; Reeves, R. Phosphorylation by Cdc2 Kinase Modulates DNA Binding Activity of High Mobility Group I Nonhistone Chromatin Protein. J. Biol. Chem. 1991, 266, 19945–19952.Google Scholar
  21. 21.
    Harrer, M.; Luhrs, H.; Bustin, M.; Scheer, U.; Hock, R. Dynamic Interaction of Hmga1a Proteins with Chromatin. J. Cell. Sci. 2004, 117, 3459–3471.CrossRefGoogle Scholar
  22. 22.
    Banks, G. C.; Li, Y.; Reeves, R. Differential In Vivo Modifications of the Hmgi(Y) Nonhistone Chromatin Proteins Modulate Nucleosome and DNA Interactions. Biochemistry 2000, 39, 8333–8346.CrossRefGoogle Scholar
  23. 23.
    Edberg, D. D.; Bruce, J. E.; Siems, W. F.; Reeves, R. In Vivo Posttranslational Modifications of the High Mobility Group A1a Proteins in Breast Cancer Cells of Differing Metastatic Potential. Biochemistry 2004, 43, 11500–11515.CrossRefGoogle Scholar
  24. 24.
    Sgarra, R.; Diana, F.; Bellarosa, C.; Dekleva, V.; Rustighi, A.; Toller, M.; Manfioletti, G.; Giancotti, V. During Apoptosis of Tumor Cells Hmga1a Protein Undergoes Methylation: Identification of the Modification Site by Mass Spectrometry. Biochemistry 2003, 42, 3575–3585.CrossRefGoogle Scholar
  25. 25.
    Edberg, D. D.; Adkins, J. N.; Springer, D. L.; Reeves, R. Dynamic and Differential In Vivo Modifications of the Isoform Hmga1a and Hmga1b Chromatin Proteins. J. Biol. Chem. 2005, 280, 8961–8973.CrossRefGoogle Scholar
  26. 26.
    Zou, Y.; Wang, Y. Tandem Mass Spectrometry for the Examination of the Post-Translational Modifications of High-Mobility Group A1 Proteins: Symmetric and Asymmetric Dimethylation of Arg25 in Hmga1a Protein. Biochemistry 2005, 44, 6293–6301.CrossRefGoogle Scholar
  27. 27.
    Sgarra, R.; Lee, J.; Tessari, M. A.; Altamura, S.; Spolaore, B.; Giancotti, V.; Bedford, M. T.; Manfioletti, G. The At-Hook of the Chromatin Architectural Transcription Factor High Mobility Group A1a Is Arginine-Methylated by Protein Arginine Methyltransferase 6. J. Biol. Chem. 2006, 281, 3764–3772.CrossRefGoogle Scholar
  28. 28.
    Jiang, X.; Wang, Y. Acetylation and Phosphorylation of High-Mobility Group A1 Proteins in Pc-3 Human Tumor Cells. Biochemistry 2006, 45, 7194–7201.CrossRefGoogle Scholar
  29. 29.
    Zou, Y.; Wang, Y. Mass Spectrometric Analysis of High-Mobility Group Proteins and Their Post-Translational Modifications in Normal and Cancerous Human Breast Tissues. J. Proteome Res. 2007, 6, 2304–2314.CrossRefGoogle Scholar
  30. 30.
    Zhang, Q.; Zhang, K.; Zou, Y.; Perna, A.; Wang, Y. A Quantitative Study on the In Vitro and In Vivo Acetylation of High Mobility Group A1 Proteins. J. Am. Soc. Mass Spectrom. 2007, 18, 1569–1578.CrossRefGoogle Scholar
  31. 31.
    Zou, Y.; Webb, K.; Perna, A. D.; Zhang, Q.; Clarke, S.; Wang, Y. A Mass Spectrometric Study on the in Vitro Methylation of Hmga1a and Hmga1b Proteins by PRMT s: Methylation Specificity, the Effect of Binding to At-Rich Duplex DNA, and the Effect of C-Terminal Phosphorylation. Biochemistry 2007, 46, 7896–7906.CrossRefGoogle Scholar
  32. 32.
    Sgarra, R.; Maurizio, E.; Zammitti, S.; Lo Sardo, A.; Giancotti, V.; Manfioletti, G. Macroscopic Differences in Hmga Oncoproteins Post-Translational Modifications: C-Terminal Phosphorylation of Hmga2 Affects Its DNA Binding Properties. J. Proteome Res. 2009, 8, 2978–2989.CrossRefGoogle Scholar
  33. 33.
    Garcia, B. A.; Shabanowitz, J.; Hunt, D. F. Characterization of Histones and Their Post-Translational Modifications by Mass Spectrometry. Curr. Opin. Chem. Biol. 2007, 11, 66–73.CrossRefGoogle Scholar
  34. 34.
    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, 4271–4280.CrossRefGoogle Scholar
  35. 35.
    Boyne, M. T.; 2nd; 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, 248–253.CrossRefGoogle Scholar
  36. 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, 240–247.CrossRefGoogle Scholar
  37. 37.
    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, 487–489.CrossRefGoogle Scholar
  38. 38.
    Reeves, R. Hmga Proteins: Isolation, Biochemical Modifications, and Nucleosome Interactions. Methods Enzymol. 2004, 375, 297–322.CrossRefGoogle Scholar
  39. 39.
    Garcia, B. A.; Mollah, S.; Ueberheide, B. M.; Busby, S. A.; Muratore, T. L.; Shabanowitz, J.; Hunt, D. F. Chemical Derivatization of Histones for Facilitated Analysis by Mass Spectrometry. Nat. Protoc. 2007, 2, 933–938.CrossRefGoogle Scholar
  40. 40.
    Plazas-Mayorca, M. D.; Zee, B. M.; Young, N. L.; Fingerman, I. M.; Leroy, G.; Briggs, S. D.; Garcia, B. A. One-Pot Shotgun Quantitative Mass Spectrometry Characterization of Histones. J. Proteome Res. 2009, 8(11), 5367–5374.CrossRefGoogle Scholar
  41. 41.
    Rappsilber, J.; Ishihama, Y.; Mann, M. Stop and Go Extraction Tips for Matrix-Assisted Laser Desorption/Ionization, Nanoelectrospray, and LC/Ms Sample Pretreatment in Proteomics. Anal. Chem. 2003, 75, 663–670.CrossRefGoogle Scholar
  42. 42.
    MacCoss, M. J.; Wu, C. C.; Yates, J. R. III. Probability-Based Validation of Protein Identifications Using a Modified SEQUEST Algorithm. Anal. Chem. 2002, 74, 5593–5599.CrossRefGoogle Scholar
  43. 43.
    Horn, D. M.; Zubarev, R. A.; McLafferty, F. W. Automated Reduction and Interpretation of High Resolution Electrospray Mass Spectra of Large Molecules. J. Am. Soc. Mass Spectrom. 2000, 11, 320–332.CrossRefGoogle Scholar
  44. 44.
    Dimaggio, P. A. Jr.; Young, N. L.; Baliban, R. C.; Garcia, B. A.; Floudas, C. A. 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. Proteom. 2009, 8(11), 2527–2543.CrossRefGoogle Scholar
  45. 45.
    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, 4093–4098.CrossRefGoogle Scholar
  46. 46.
    Wisniewski, J. R.; Zougman, A.; Mann, M. Nepsilon-Formylation of Lysine Is a Widespread Post-Translational Modification of Nuclear Proteins Occurring at Residues Involved in Regulation of Chromatin Function. Nucleic Acids Res. 2008, 36, 570–577.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2010

Authors and Affiliations

  • Nicolas L. Young
    • 1
  • Mariana D. Plazas-Mayorca
    • 2
  • Peter A. DiMaggio
    • 3
  • Ian Z. Flaniken
    • 1
  • Andrea J. Beltran
    • 1
  • Neeli Mishra
    • 1
  • Gary LeRoy
    • 1
  • Christodoulos A. Floudas
    • 3
  • Benjamin A. Garcia
    • 1
    • 2
  1. 1.Department of Molecular BiologyPrinceton UniversityPrincetonUSA
  2. 2.Department of ChemistryPrinceton UniversityPrincetonUSA
  3. 3.Department of Chemical EngineeringPrinceton UniversityPrincetonUSA

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