Peptide Sequencing by Nanoelectrospray Tandem Mass Spectrometry

  • Ole Nørregaard Jensen
  • Matthias Wilm
Part of the Springer Protocols Handbooks book series (SPH)


Electrospray ionization mass spectrometry (ESI-MS) (1) has had a profound influence on biological research over the last decade. With this technique it is possible to generate and characterize gas-phase analyte ions from aqueous solutions of proteins, peptides, and other classes of biomolecules. ESI is performed at atmospheric pressure, which simplifies sample preparation and handling and allows on-line coupling of chromatography, such as capillary high-performance liquid chromatography (HPLC), to mass spectrometers (LC-MS). The use of ESI in combination with tandem mass spectrometry (MS/MS) provides the capability for amino acid sequencing of peptides. The optimization and miniaturization of peptide sample preparation methods for ESI as well as the development of highly sensitive tandem mass spectrometers, such as triple quadrupoles, ion traps, and quadrupole-time-of-flight (Q-TOF) hybrid instruments, makes it possible and almost routine to obtain amino acid sequences from subpicomole levels of protein in many laboratories. Peptide sequencing is typically performed by nanoelectrospray MS/MS analysis of crude, concentrated peptide mixtures or by hyphenated techniques, such as capillary HPLC coupled to micro/ nanoelectrospray-MS/MS. The sets of peptide tandem mass spectra generated in such experiments are used to query biological sequence databases with the aim to identify all protein components present in the sample. In this chapter we describe practical aspects of nanoelectrospray mass spectrometry aimed at amino acid sequencing of peptides at subpicomole levels. We do not consider LC-MS/MS, although many of the features of the two analytical approaches are very similar.


Tryptic Peptide Tandem Mass Spectrum Chromatographic Resin Nanoelectrospray Mass Spectrometry Peptide Tandem Mass Spectrum 
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Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Ole Nørregaard Jensen
    • 1
  • Matthias Wilm
    • 2
  1. 1.Department of Biochemistry and Molecular BiologyOdense UniversityDenmark
  2. 2.Department of Biochemistry and Molecular BiologyOdense UniversityDenmark

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