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)	456
Venue 2-year citedness	1.63

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, University of California San Diego
2. Igor V. Chernushevich · PE 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
7. Matthias Mann · European Molecular Biology Laboratory, Max Planck Institute of Biochemistry

Methods and tools

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

Cited by (9)

• Quantification of snake venom proteomes by mass spectrometry - considerations and perspectives (2023) both
• De Novo Sequencing and Homology Searching (2012) both
• Dimethyl isotope labeling assisted de novo peptide sequencing (2010) crossref
• De novo sequencing of peptides by MS/MS (2010) crossref
• De Novo Sequencing of Neuropeptides Using Reductive Isotopic Methylation and Investigation of ESI QTOF MS/MS Fragmentation Pattern of Neuropeptides with N-Terminal Dimethylation (2005) crossref
• De Novo Peptide Sequencing Based on a Divide-and-Conquer Algorithm and Peptide Tandem Spectrum Simulation (2004) crossref
• “De novo” peptide sequencing by MALDI-quadrupole-ion trap mass spectrometry: A preliminary study (2003) crossref
• De novo peptide sequencing and quantitative profiling of complex protein mixtures using mass-coded abundance tagging (2002) crossref
• De novo peptide sequencing via tandem mass spectrometry (1999) crossref

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