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Related Experiment Videos

Absolute multiplexed quantitative analysis of protein expression during muscle development using QconCAT.

Jenny Rivers1, Deborah M Simpson, Duncan H L Robertson

  • 1Proteomics and Functional Genomics Group, Faculty of Veterinary Science, University of Liverpool, Liverpool L69 7ZJ, United Kingdom.

Molecular & Cellular Proteomics : MCP
|May 19, 2007
PubMed
Summary
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Quantifying multiple proteins simultaneously is now possible with QconCATs (quantitativeconcatemer proteins). This method uses engineered proteins containing multiple proteotypic peptides for accurate, cost-effective absolute protein quantification in proteomics.

Area of Science:

  • Proteomics
  • Biotechnology
  • Analytical Chemistry

Background:

  • Absolute protein quantification in proteomics typically requires synthesizing unique stable isotope-labeled peptides for each target protein, which is costly and time-consuming.
  • Multiplexing protein quantification is desirable for high-throughput and comprehensive proteomic analyses.

Purpose of the Study:

  • To develop and validate a novel method for simultaneous absolute quantification of multiple proteins using engineered artificial proteins called QconCATs.
  • To define the characteristics, error sources, and statistical behavior of QconCAT-based analyses.

Main Methods:

  • Designed and expressed QconCAT proteins, concatenating proteotypic peptides from target proteins, in E. coli using stable isotope labeling.
  • Purified and quantified the labeled QconCAT protein, then used it as an internal standard in homogenized chicken muscle samples.

Related Experiment Videos

  • Analyzed peptides using liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (LC-ESI-TOF MS) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS).

    • Main Results:

    • Demonstrated successful expression and labeling of QconCAT proteins containing peptides from 20 target proteins.
    • Confirmed that QconCATs are rapidly digested by trypsin, enabling their use as internal standards for absolute quantification.
    • Assessed technical variance and compared it with biological variance, validating the method's precision.

    Conclusions:

    • QconCATs provide an efficient and precise method for simultaneous absolute quantification of multiple proteins, subproteomes, or entire proteomes.
    • This approach significantly reduces the cost and effort associated with traditional single-protein quantification methods in proteomics.