Rapid de novo peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer

peer-reviewed · Rapid Communications in Mass Spectrometry · 1997

peer-reviewed · Rapid Communications in Mass Spectrometry · 1997. Andrej Shevchenko et al. Protein microanalysis usually involves the sequencing of gel-separated proteins available in very small…
Date 1997-06-15
Type peer-reviewed
Venue Rapid Communications in Mass Spectrometry
Publisher Wiley
Contribution algorithm
DOI 10.1002/(SICI)1097-0231(19970615)11:9<1015::AID-RCM958>3.0.CO;2-H
Citations (OpenAlex) 455
Venue 2-year citedness 1.80

Abstract

Protein microanalysis usually involves the sequencing of gel-separated proteins available in very small amounts. While mass spectrometry has become the method of choice for identifying proteins in databases, in almost all laboratories ‘de novo’ protein sequencing is still performed by Edman degradation. Here we show that a combination of the nanoelectrospray ion source, isotopic end labeling of peptides and a quadrupole/ time-of-flight instrument allows facile read-out of the sequences of tryptic peptides. Isotopic labeling was performed by enzymatic digestion of proteins in 1:1 16O/18O water, eliminating the need for peptide derivatization. A quadrupole/time-of-flight mass spectrometer was constructed from a triple quadrupole and an electrospray time-of-flight instrument. Tandem mass spectra of peptides were obtained with better than 50 ppm mass accuracy and resolution routinely in excess of 5000. Unique and error tolerant identification of yeast proteins as well as the sequencing of a novel protein illustrate the potential of the approach. The high data quality in tandem mass spectra and the additional information provided by the isotopic end labeling of peptides enabled automated interpretation of the spectra via simple software algorithms. The technique demonstrated here removes one of the last obstacles to routine and high throughput protein sequencing by mass spectrometry.

Authors

  1. Andrej Shevchenko · European Molecular Biology Laboratory, Max Planck Institute of Molecular Cell Biology and Genetics, Max Planck Society, University of California San Diego
  2. Igor V. Chernushevich · Max Planck Institute of Molecular Cell Biology and Genetics, Max Planck Society, PE SCIEX, SCIEX, University of Manitoba
  3. Werner Ens · University of Manitoba
  4. Kenneth G. Standing · University of Manitoba
  5. Bruce A. Thomson · PE SCIEX
  6. Matthias Wilm · European Molecular Biology Laboratory, Max Planck Institute of Molecular Cell Biology and Genetics, University of Southern Denmark
  7. Matthias Mann · European Molecular Biology Laboratory, Max Planck Institute of Biochemistry, Max Planck Institute of Molecular Cell Biology and Genetics, Novo Nordisk Foundation, University of California San Diego, University of Copenhagen, University of Southern Denmark

Methods and tools

  • Rapid QTOF de novo sequencing: Nanoelectrospray, isotopic-labeling, and quadrupole/time-of-flight workflow for rapid de novo peptide sequencing.

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