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Related Experiment Videos

Quantitative proteome analysis using isotope-coded affinity tags and mass spectrometry.

Yuzuru Shiio1, Ruedi Aebersold

  • 1Children's Cancer Research Institute, The University of Texas Health Science Center, San Antonio, Texas 78229, USA.

Nature Protocols
|April 5, 2007
PubMed
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Isotope-coded affinity tags (ICAT) and tandem mass spectrometry enable precise protein identification and quantification in complex biological samples. This method advances proteomics by analyzing global and subproteome expression changes, even for challenging proteins.

Area of Science:

  • Proteomics
  • Biochemistry
  • Analytical Chemistry

Background:

  • Systematic identification and quantification of proteins are crucial objectives in proteomics research.
  • Analyzing complex biological mixtures like proteomes, tissues, and body fluids presents significant challenges.
  • Existing gel-based proteomics technologies have limitations in analyzing certain proteins.

Purpose of the Study:

  • To describe a protocol for protein identification and quantification using isotope-coded affinity tags (ICAT) and tandem mass spectrometry.
  • To enable accurate quantitative analysis of proteins in various biological samples, including complex mixtures and subproteomes.
  • To provide a method suitable for both large-scale and small-scale proteomic analyses.

Main Methods:

  • Protein-sample labeling using ICAT reagents.

Related Experiment Videos

  • Chromatographic fractionation of ICAT-labeled tryptic peptides.
  • Protein identification and quantification via tandem mass spectrometry.

    • Main Results:

    • Successful application of ICAT and tandem mass spectrometry for protein identification and quantification in complex mixtures.
    • Demonstrated ability to analyze global protein expression changes, protein alterations in subcellular fractions, protein complexes, secretion, and body fluids.
    • Enabled quantitative analysis of proteins, including those recalcitrant to gel-based methods.

    Conclusions:

    • The described ICAT-based protocol offers a robust approach for comprehensive proteomic analysis.
    • This method significantly enhances the ability to identify and quantify proteins across diverse biological contexts.
    • It provides a valuable tool for advancing quantitative proteomics research, overcoming limitations of previous technologies.