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Absolute two-point quantification of proteins using dimethylated proteotypic peptides.

The Trung Tran1, Ravi Chand Bollineni1, Christian Jeffrey Koehler1

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This study introduces a cost-effective two-point quantification method using dual stable isotopes for absolute protein quantification in proteomics. The approach enhances data reliability and offers internal quality control for accurate protein concentration measurements.

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Absolute targeted proteomics commonly uses synthetic stable isotopically labeled peptides for protein concentration determination via LC-MS.
  • Existing methods can be costly and may benefit from more efficient quantification strategies.

Purpose of the Study:

  • To evaluate the use of two different stable isotopes of the same peptide as a spike-in for absolute quantification.
  • To develop a cost-effective and reliable method for absolute protein quantification.

Main Methods:

  • Peptide labeling via reductive amination to generate light, medium, and heavy isotopic forms.
  • Application of a two-point quantification method using dual stable isotope-labeled spike-in peptides.
  • Analysis of apolipoprotein A-I, apolipoprotein B-100, and leucine-rich alpha-2-glycoprotein in serum samples.

Main Results:

  • Accurate measurement of endogenous protein concentrations across four orders of magnitude in serum.
  • Achieved coefficient of variation (CV) below 20% and high correlation (R² = 0.99) across replicates.
  • Demonstrated internal quality control capability by detecting methodical errors through ratio deviations.

Conclusions:

  • The two-point quantification method using dual stable isotopes provides a reliable and cost-effective alternative for absolute protein quantification.
  • This approach offers significant cost savings compared to traditional methods using synthetic stable isotopically labeled peptides.
  • The internal quality control feature enhances the robustness of protein concentration measurements, particularly for multiplexed analyses.