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The QUEST system for quantitative analysis of two-dimensional gels.

J I Garrels1

  • 1Cold Spring Harbor Laboratory, New York 11724.

The Journal of Biological Chemistry
|March 25, 1989
PubMed
Summary

The QUEST system accurately quantifies radiolabeled proteins using two-dimensional gel electrophoresis and pattern matching. This advanced analysis enables precise protein detection and database creation for cell studies.

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Area of Science:

  • Biochemistry and Molecular Biology
  • Proteomics
  • Bioinformatics

Background:

  • Two-dimensional gel electrophoresis (2D-PAGE) is a powerful technique for separating complex protein mixtures.
  • Accurate quantification and matching of protein spots across multiple gels are crucial for quantitative proteomics.
  • Existing methods often face challenges with spot resolution, matching accuracy, and data consistency.

Purpose of the Study:

  • To describe the strategies and methods of the QUEST system for analyzing two-dimensional gels.
  • To evaluate the performance and accuracy of the QUEST system for protein quantification and pattern matching.
  • To establish a quantitative protein database for normal and transformed REF52 cells.

Main Methods:

  • Proteins are resolved on 2D gels and detected using calibrated film exposures.
  • Quantification is performed in disintegrations per minute (DPM) or as a fraction of total protein radioactivity.
  • Two-dimensional Gaussian fitting is used for spot quantitation and resolving overlapping spots.
  • Pattern matching is conducted on groups of gels (matchsets) with automated spot addition for enhanced accuracy.
  • Spot fitting is repeated post-matching to refine results.

Main Results:

  • The QUEST system achieves high matching rates, with up to 97% of spots matched per pattern and less than 1% inconsistently matched.
  • Approximately 2000 proteins are detected per gel, with 1600 identified as high-quality spots.
  • The average coefficient of variation for high-quality spot quantitation is 21%.
  • Detected protein intensities range from 4 to 20,000 ppm of total protein synthesis.

Conclusions:

  • The QUEST system provides a robust and accurate method for quantitative analysis of 2D gel electrophoresis data.
  • The system significantly enhances spot detection and matching accuracy, enabling reliable proteomic studies.
  • QUEST facilitates the creation of quantitative protein databases, as demonstrated with REF52 cell lines.

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