Protocol

2-D Proteome Analysis Protocols

Volume 112 of the series Methods in Molecular Biology pp 571-588

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

  • Ole N. JensenAffiliated withProtein Research Group, Department of Molecular Biology, Odense University
  • , Matthias WilmAffiliated withProtein and Peptide Group, European Molecular Biology Laboratory
  • , Andrej ShevchenkoAffiliated withProtein and Peptide Group, European Molecular Biology Laboratory
  • , Matthias MannAffiliated withProtein and Peptide Group, European Molecular Biology Laboratory

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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 (24). 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 (57), 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.