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

Journal of the American Society for Mass Spectrometry volume 21, pages 960–970 (2010)Cite this article

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.

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Authors and Affiliations

  1. Department of Molecular Biology, Princeton University, 08544, Princeton, NJ, USA

    Nicolas L. Young, Ian Z. Flaniken, Andrea J. Beltran, Neeli Mishra, Gary LeRoy & Benjamin A. Garcia

  2. Department of Chemistry, Princeton University, Princeton, New Jersey, USA

    Mariana D. Plazas-Mayorca & Benjamin A. Garcia

  3. Department of Chemical Engineering, Princeton University, Princeton, New Jersey, USA

    Peter A. DiMaggio & Christodoulos A. Floudas

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  1. Nicolas L. Young
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  2. Mariana D. Plazas-Mayorca
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  3. Peter A. DiMaggio
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  4. Ian Z. Flaniken
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  6. Neeli Mishra
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  8. Christodoulos A. Floudas
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Additional information

This publication is dedicated to Professor Neil L. Kelleher, the 2009 Biemann Medal award recipient, whose continued dedication and extensive research work have propagated mass spectrometry studies of intact proteins and scientific careers thereof world-wide.

These authors contributed equally to this work.

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Young, N.L., Plazas-Mayorca, M.D., DiMaggio, P.A. et al. Collective mass spectrometry approaches reveal broad and combinatorial modification of high mobility group protein A1a. J Am Soc Mass Spectrom 21, 960–970 (2010). https://doi.org/10.1016/j.jasms.2010.01.020

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  • DOI: https://doi.org/10.1016/j.jasms.2010.01.020

Keywords

  • Mass Spectrometry Analysis
  • High Mobility Group
  • HMGA Protein
  • Propionic Anhydride
  • Grow HeLa Cell