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Related Experiment Videos

18O labeling: a tool for proteomics.

I I Stewart1, T Thomson, D Figeys

  • 1MDS-Proteomics Inc., 251 Attwell Drive, Toronto, Ontario M9W 7H4, Canada. istewart@mdsp.com

Rapid Communications in Mass Spectrometry : RCM
|December 18, 2001
PubMed
Summary
This summary is machine-generated.

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This study presents a novel 18O labeling method for protein quantification in diagnostics. The developed protocol optimizes peptide labeling efficiency and minimizes sample loss for accurate proteomics analysis.

Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biotechnology

Background:

  • Accurate protein quantification is crucial for diagnostic applications.
  • Existing methods for protein labeling and quantification have limitations.
  • Oxygen isotope labeling (18O) offers a promising approach for relative protein quantitation.

Purpose of the Study:

  • To evaluate the use of trypsin and 18O-enriched water for proteolytic labeling and protein quantification.
  • To develop and optimize a protocol for efficient and reliable 18O labeling of peptides.
  • To assess factors affecting labeling efficiency and sample recovery in proteomics workflows.

Main Methods:

  • Proteolytic digestion using trypsin with 18O-enriched water.
  • Development of a protocol to prevent peptide back-exchange during labeling.

Related Experiment Videos

  • Evaluation of labeling efficiency using varying 16O/18O buffer ratios.
  • Assessment of sample recovery using Ziptip and Speedvac methods.
  • Analysis of in-solution digestion efficiency as a function of protein concentration.

    • Main Results:

    • Comparative or relative protein quantitation can be effectively performed using 18O labeling.
    • A protocol was developed to conserve labeled peptides without fear of back-exchange under specific pH conditions.
    • Peptide labeling efficiency follows a probability-based y = x(2) relationship, deviating from linear expectations.
    • Sample recovery is reduced at low peptide concentrations, and adsorptive losses occur during Speedvac drying.
    • In-solution digestion efficiency decreases linearly with decreasing protein concentration, highlighting potential quantitation errors.

    Conclusions:

    • 18O labeling is a powerful tool for optimizing proteomics process development.
    • The developed protocol enables accurate relative protein quantitation for diagnostic purposes.
    • Understanding factors affecting labeling efficiency and sample recovery is critical for reliable proteomics data.