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Related Experiment Video

Updated: May 1, 2026

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water
09:43

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Quantitative protein analysis using enzymatic [¹⁸O]water labeling.

Mary Joan Castillo1, Kristy J Reynolds2, Alexander Gomes1

  • 1Department of Chemistry, University of Connecticut, Storrs, Connecticut.

Current Protocols in Protein Science
|April 3, 2014
PubMed
Summary

This study details a method for differential oxygen isotope labeling of peptides using immobilized trypsin. This technique enables accurate relative protein quantification in comparative proteomics via mass spectrometry.

Keywords:
18O-labelingback-exchangeenzymatic oxygen labelingimmobilized trypsinquantitative proteomics

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Last Updated: May 1, 2026

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Proteomics

Background:

  • Comparative proteomics is crucial for understanding biological processes.
  • Accurate quantification of relative protein levels is essential for identifying biomarkers and elucidating signaling pathways.
  • Existing methods for protein quantification have limitations in sensitivity and scope.

Purpose of the Study:

  • To describe a detailed protocol for differential oxygen isotope labeling of peptides.
  • To present a method for mass spectrometric quantification of relative protein levels using this labeling technique.
  • To discuss experimental considerations for effective oxygen label incorporation and stabilization.

Main Methods:

  • Enzymatic oxygen exchange of tryptic peptides using immobilized trypsin to label the peptide C-terminus with oxygen isotopes.
  • Differential labeling using oxygen-18 ([¹⁸O]) and oxygen-16 ([¹⁶O]) isotopes.
  • Mass spectrometric analysis for quantifying peptides based on their differential isotopic signatures.

Main Results:

  • Successful implementation of stepwise oxygen isotope labeling at the peptide C-terminus.
  • Demonstration of mass spectrometric quantification of differentially labeled peptides.
  • Identification of key experimental factors influencing label incorporation and stability.

Conclusions:

  • The described method provides a robust approach for differential oxygen isotope labeling of peptides.
  • This technique facilitates accurate relative protein quantification in comparative proteomic studies.
  • The protocol offers valuable insights for researchers employing isotopic labeling strategies in proteomics.