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DOSCATs: Double standards for protein quantification.

Richard J Bennett1, Deborah M Simpson1, Stephen W Holman1

  • 1Centre for Proteome Research, Institute of Integrative Biology, Biosciences Building, University of Liverpool, Liverpool L69 7ZB, UK.

Scientific Reports
|April 4, 2017
PubMed
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We developed novel calibration standards, DOSCATs (DOuble Standard conCATamers), to unify mass spectrometry (MS) and western blotting (WB) for absolute protein quantification. These standards enable seamless, orthogonal protein quantification across both techniques.

Area of Science:

  • Proteomics
  • Molecular Biology
  • Biochemistry

Background:

  • Absolute protein quantification is crucial but challenging, with mass spectrometry (MS) and western blotting (WB) offering complementary strengths.
  • MS provides high data quality and reproducibility, while WB offers greater sensitivity and accessibility.
  • Current workflows rarely integrate MS and WB for orthogonal quantification.

Purpose of the Study:

  • To introduce DOSCATs (DOuble Standard conCATamers), a novel calibration standard technology.
  • To enable the simultaneous use of MS and WB for absolute protein quantification within a unified workflow.
  • To facilitate seamless quantitative transitions between MS and WB.

Main Methods:

  • Design and construction of a DOSCAT protein incorporating epitope sequences and tryptic peptides.

Related Experiment Videos

  • Development of DOSCATs based on QconCAT technology for dual-purpose calibration.
  • Application of DOSCATs for quantifying five proteins in the NF-κB pathway.

    • Main Results:

    • DOSCATs function as multiplexed, dual-purpose standards for both MS and WB.
    • Excellent agreement was observed between MS and WB measurements for protein fold change and absolute copy per cell values for three target proteins.
    • Demonstrated seamless quantitative transition from MS to WB using a single workflow.

    Conclusions:

    • DOSCATs effectively bridge MS and WB platforms for orthogonal protein quantification.
    • This technology enhances the reliability and accessibility of absolute protein quantification.
    • DOSCATs represent a significant advancement for integrated proteomic analysis.