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Related Experiment Videos

Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis.

M B Smolka1, H Zhou, S Purkayastha

  • 1Departamento de Bioquímica, Universidade Estadual de Campinas, Campinas, Sao Paulo, Brazil.

Analytical Biochemistry
|September 25, 2001
PubMed
Summary

Isotope Coded Affinity Tag (ICAT) reagents combined with tandem mass spectrometry offer a new quantitative proteomics method. Optimized labeling protocols ensure specific and reproducible protein quantification in complex mixtures.

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Quantitative proteomics is essential for understanding cellular processes.
  • Existing methods face challenges in specificity and reproducibility.
  • Isotope Coded Affinity Tag (ICAT) reagents offer a promising approach for quantitative analysis.

Purpose of the Study:

  • To systematically evaluate and optimize the labeling protocol for ICAT reagents.
  • To ensure specificity, quantitation, generality, robustness, and reproducibility of the ICAT method.
  • To demonstrate the efficacy of optimized ICAT labeling in complex biological samples.

Main Methods:

  • Site-specific, covalent labeling of proteins using ICAT reagents (isotopically normal or heavy).
  • Proteolysis of labeled protein mixtures.

Related Experiment Videos

  • Isolation and tandem mass spectrometry analysis of labeled peptides.
  • Systematic evaluation of ICAT reagent concentration, protein, SDS, urea concentrations, and reaction time.

    • Main Results:

    • Optimized labeling conditions were established for ICAT reagents.
    • Specific and quantitative protein labeling was achieved.
    • The method demonstrated robustness and reproducibility across various conditions.
    • Successful application of optimized ICAT labeling to standard proteins and complex yeast cell lysates.

    Conclusions:

    • The optimized ICAT reagent labeling protocol is specific, quantitative, and reproducible.
    • This method provides a reliable approach for quantitative proteomics.
    • The optimized ICAT method is applicable to complex biological samples, advancing proteomic research.