2-D Proteome Analysis Protocols pp 571-588

Part of the Methods in Molecular Biology book series (MIMB, volume 112)

Peptide Sequencing of 2-DE Gel-Isolated Proteins by Nanoelectrospray Tandem Mass Spectrometry

  • Ole N. Jensen
  • Matthias Wilm
  • Andrej Shevchenko
  • Matthias Mann

Abstract

Shortly after the introduction of electrospray as a viable ionization technique for large molecules (1), electrospray tandem mass spectrometry (ES MS/MS) techniques, such as HPLC-ES MS/MS, were used successfully for peptide sequencing at picomole and subpicomole levels (2, 3, 4). In LC-MS/MS, peptide sequence information is generated during the short time, 10–30 s, that a peptide elutes from the HPLC column run at a flow rate of 0.5–5 μL/min. This time frame rarely allows optimization of experimental parameters for MS/MS sequencing of individual peptides unless several LC-MS/MS experiments can be performed on a sample. With the introduction of the nanoelectrospray ion source (5, 6, 7), the time constraint of tandem mass spectrometry has been removed, and peptide sequencing has been reliably extended to the femtomole level of gel-isolated protein.

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Copyright information

© Humana Press Inc., Totowa, NJ 1999

Authors and Affiliations

  • Ole N. Jensen
    • 1
  • Matthias Wilm
    • 2
  • Andrej Shevchenko
    • 2
  • Matthias Mann
    • 2
  1. 1.Protein Research Group, Department of Molecular BiologyOdense UniversityOdenseDenmark
  2. 2.Protein and Peptide GroupEuropean Molecular Biology LaboratoryHeidelbergGermany

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