Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry (original) (raw)

Nature volume 379, pages 466–469 (1996)Cite this article

Abstract

MOLECULAR analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry1 has been applied to the high-sensitivity sequencing of short peptides2, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electro-phoresis, using nano-electrospray3,4 tandem mass spectrometry5,6. As little as 5ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells _in vitro_and thus may have potential antiangiogenic effects on solid tumours.

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Author notes

  1. Lothar Schweigerer
    Present address: Children's Hospital, University of Marburg, Deutschhausstrasse 12, 35033, Marburg, Germany
  2. Theodore Fotsis
    Present address: Laboratory of Biological Chemistry, University of loannina Medical School, GR 45110, loannina, Greece
  3. Matthias Mann: To whom correspondence should be addressed.

Authors and Affiliations

  1. Protein & Peptide Group, EMBL, Meyerhofstrasse 1, 69117, Heidelberg, Germany
    Matthias Wilm, Andrej Shevchenko, Tony Houthaeve & Matthias Mann
  2. Division of Oncology and Haematology, Children's Hospital, University of Heidelberg, INF 150, 69120, Heidelberg, Germany
    Stephen Breit, Lothar Schweigerer & Theodore Fotsis

Authors

  1. Matthias Wilm
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  2. Andrej Shevchenko
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  3. Tony Houthaeve
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  4. Stephen Breit
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  5. Lothar Schweigerer
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  6. Theodore Fotsis
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  7. Matthias Mann
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Wilm, M., Shevchenko, A., Houthaeve, T. et al. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry.Nature 379, 466–469 (1996). https://doi.org/10.1038/379466a0

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