Research Article

Nanocapillary liquid chromatography interfaced to tandem matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry: Mapping the nuclear proteome of human fibroblasts

  1. Johan Malmström1,
  2. Kristoffer Larsen1,
  3. Lars Malmström2,
  4. Ellen Tufvesson1,
  5. Ken Parker3,
  6. Jason Marchese3,
  7. Brian Williamson3,
  8. Dale Patterson3,
  9. Steve Martin3,
  10. Peter Juhasz3,
  11. Gunilla Westergren-Thorsson1,
  12. György Marko-Varga4,*

Article first published online: 6 NOV 2003

DOI: 10.1002/elps.200305619

Issue

ELECTROPHORESIS

ELECTROPHORESIS

Special Issue: Paper Symposium Miniaturization 2003

Volume 24, Issue 21, pages 3806–3814, No. 21 November 2003

Additional Information

How to Cite

Malmström, J., Larsen, K., Malmström, L., Tufvesson, E., Parker, K., Marchese, J., Williamson, B., Patterson, D., Martin, S., Juhasz, P., Westergren-Thorsson, G. and Marko-Varga, G. (2003), Nanocapillary liquid chromatography interfaced to tandem matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry: Mapping the nuclear proteome of human fibroblasts. ELECTROPHORESIS, 24: 3806–3814. doi: 10.1002/elps.200305619

Author Information

  1. 1

    Cell & Molecular Biology, BMC, C13, University of Lund, Lund, Sweden

  2. 2

    Department of Electrical Measurement, Lund Institute of Technology, Lund, Sweden

  3. 3

    Applied Biosystems, Framingham, MA, USA

  4. 4

    Department of Analytical Chemistry, University of Lund, Lund, Sweden

*University of Lund, P. O. Box 127 SE-221 00 Lund, Sweden Fax: +46-46-222-4544

Publication History

  1. Issue published online: 6 NOV 2003
  2. Article first published online: 6 NOV 2003
  3. Manuscript Received: 27 JUL 2003

SEARCH

SEARCH BY CITATION

ARTICLE TOOLS

Get PDF (312K)

Keywords:

  • Electrospray ionization-tandem mass spectrometry;
  • Fibroblast;
  • Matrix-assisted laser desorption/ionization-tandem mass spectrometry;
  • Miniaturization;
  • Nanocapillary liquid chromatography;
  • Nucleus

Abstract

Miniaturized liquid chromatography nanoseparation in combination with minigel fractionation of human primary cell nuclei is presented. We obtained high-sensitivity and high-throughput identification of expressed proteins by subcellular fractionation and nanocapillary liquid chromatography interfaced to both electrospray ionization (ESI)- and matrix-assisted laser desorption/ionisation (MALDI) tandem mass spectrometry. The reversed-phase nanocapillary eluents were applied directly onto the MALDI target plate as discrete crystal spots using in-line matrix infusion. When working with primary cells, only a limited amount of sample is available. To maximize the number of identified proteins from a restricted amount of sample, miniaturized sample preparation protocols and nanoflow separation is a necessity, especially when working with low-abundant proteins. From the same isolated nuclear sample, complementary separation of intact proteins by two-dimensional (2-D) gel electrophoresis was made. In total 594 gene products from the nuclear preparations were identified out of which 261 were unique. Several proteins involved in transcriptional events were identified such as TATA-binding protein, EBNA-co-activator, and interleukin enhancer binding proteins, indicating that sufficient proteomic depth is obtained to study transcriptional controlling events. Our results suggest that by sample prefractionation and downscaled nanoflow separation along with a combined mass spectrometry strategy, it is possible to identify a large number of nuclear proteins from human primary cells. These findings are of particular importance due to the disease link of these targets cells.

Get PDF (312K)