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Related Experiment Videos

Absolute quantification of specific proteins in complex mixtures using visible isotope-coded affinity tags.

Yu Lu1, Patricia Bottari, Frantisek Turecek

  • 1Departments of Chemistry and Biochemistry, University of Washington, Seattle, Washington 98195, USA.

Analytical Chemistry
|July 16, 2004
PubMed
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We developed Visible Isotope-Coded Affinity Tag (VICAT) reagents for quantifying proteins in complex samples. This method precisely measured human group V phospholipase A(2) in lung macrophages, outperforming Western blots.

Area of Science:

  • Proteomics
  • Biochemistry
  • Analytical Chemistry

Background:

  • Quantitative protein identification in complex biological mixtures is crucial for biological and medical research.
  • Existing methods often lack the precision or sensitivity required for absolute protein quantification.

Purpose of the Study:

  • To introduce and validate a novel protein tagging reagent, Visible Isotope-Coded Affinity Tag (VICAT), for absolute protein quantification.
  • To demonstrate the application of VICAT reagents in determining the abundance of a specific protein in a complex biological sample.

Main Methods:

  • VICAT reagents were designed to tag cysteine residues or thioacetylated amino groups, incorporating a biotin affinity handle, a visible moiety, a photocleavable linker, and an isotope tag.
  • Peptides were analyzed using isoelectric focusing followed by micro-liquid chromatography/electrospray ionization mass spectrometry in selected reaction monitoring mode.

Related Experiment Videos

  • Absolute quantification of human group V phospholipase A(2) in eukaryotic cell lysates was performed.

    • Main Results:

    • The VICAT method successfully quantified human group V phospholipase A(2) in human lung macrophages at 66 fmol per 100 μg of cell protein.
    • Western blot analysis yielded inconclusive data for the same protein, highlighting the sensitivity of the VICAT approach.
    • The visible moiety allowed for chromatographic tracking independent of mass spectrometry.

    Conclusions:

    • VICAT reagents provide a robust method for absolute protein quantification in complex biological samples.
    • This technology offers advantages over traditional methods like Western blotting for sensitive and accurate protein abundance determination.
    • VICAT reagents hold significant potential for applications such as biomarker discovery and validation in clinical samples like serum.