Technology

Free-flow electrophoresis for top-down proteomics by Fourier transform ion cyclotron resonance mass spectrometry

  1. Séverine A. Ouvry-Patat1,†,
  2. Matthew P. Torres1,†,
  3. Hung-Hiang Quek2,3,
  4. Craig A. Gelfand2,
  5. Patrick O'Mullan2,
  6. Mikkel Nissum4,
  7. Gottfried K. Schroeder1,
  8. Jun Han5,
  9. Monica Elliott5,
  10. Deanna Dryhurst5,
  11. Juan Ausio5,
  12. Richard Wolfenden1,
  13. Christoph H. Borchers Dr.1,5,*

Article first published online: 23 JUL 2008

DOI: 10.1002/pmic.200800079

Issue

PROTEOMICS

PROTEOMICS

Volume 8, Issue 14, pages 2798–2808, No. 14 July 2008

Additional Information

How to Cite

Ouvry-Patat, S. A., Torres, M. P., Quek, H.-H., Gelfand, C. A., O'Mullan, P., Nissum, M., Schroeder, G. K., Han, J., Elliott, M., Dryhurst, D., Ausio, J., Wolfenden, R. and Borchers, C. H. (2008), Free-flow electrophoresis for top-down proteomics by Fourier transform ion cyclotron resonance mass spectrometry. PROTEOMICS, 8: 2798–2808. doi: 10.1002/pmic.200800079

Author Information

  1. 1

    Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, NC, USA

  2. 2

    BD Diagnostics, Preanalytical Systems, Franklin Lakes, NJ, USA

  3. 3

    Temasek Applied Science School, Temasek Polytechnic, Singapore

  4. 4

    BD Diagnostics, Martinsried, Germany

  5. 5

    Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada

  1. †

    These authors contributed equally to this work.

*Genome BC Proteomics Center, University of Victoria, 3101-4464 Markham St., Victoria, BC, Canada V8Z 7X8 Fax: +1-250-483-3238

Publication History

  1. Issue published online: 23 JUL 2008
  2. Article first published online: 23 JUL 2008
  3. Manuscript Received: 28 JAN 2008

Funded by

  • Cystic Fibrosis Foundation. Grant Number: CFFTIBORCHE05U0
  • CDC. Grant Number: H75/CCH424675
  • BD (Becton Dickinson) Biosciences
  • North Carolina Biotechnology Center. Grant Number: NCBC 2005-IDG-1015
  • National Institute of Health. Grant Number: 1-S10-RR019889-01

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Keywords:

  • Acetylation;
  • Free-flow electrophoresis (FFE);
  • FTICR-MS;
  • Histone H2A-IV;
  • Top-down proteomics

Abstract

High-efficiency prefractionation of complex protein mixtures is critical for top-down proteomics, i.e., the analysis of intact proteins by MS. Free-flow electrophoresis (FFE) can be used for IEF to separate proteins within a pH gradient according to their pIs. In an FFE system, this separation is performed entirely in the liquid phase, without the need for particulate chromatographic media, gels, or membranes. Herein, we demonstrated the compatibility of IEF-FFE with ESI-Fourier transform ICR MS (ESI-FTICR-MS) for top-down experiments. We demonstrated that IEF-FFE of intact proteins were highly reproducible between FFE instruments, between laboratories, and between analyses. Applying native (0.2% hydroxypropylmethyl cellulose) IEF-FFE to an enzyme resulted in no decrease in enzyme activity; applying either native or denaturing (8 M urea) IEF-FFE to a four-protein mixture with different pIs resulted in isolation of each protein into separate fractions in a 96-well plate. After desalting, each protein was sequenced by top-down MS/MS. As an application of this technique, chicken erythrocyte histone H2A-IV and its major modified forms were enriched by IEF-FFE. Top-down analysis revealed Lys-5 to be a major acetylation site, in addition to N-terminal acetylation.

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