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Absolute quantitation of protein posttranslational modification isoform.

Zhu Yang1, Ning Li

  • 1Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China, zhuyang@ust.hk.

Methods in Molecular Biology (Clifton, N.J.)
|May 2, 2015
PubMed
Summary

Accurately quantify protein isoforms using the Absolute Quantitation of Isoforms of Posttranslationally Modified Proteins (AQUIP) method. This technique uses stable isotope-labeled peptides and mass spectrometry to determine absolute protein amounts and posttranslational modification occupancy.

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Mass spectrometry is crucial for protein characterization and quantification in biological samples.
  • Accurate protein quantification is essential for mathematical modeling of biological systems.
  • Quantitative study of posttranslational modifications (PTMs) is vital for understanding protein function.

Purpose of the Study:

  • To present the Absolute Quantitation of Isoforms of Posttranslationally Modified Proteins (AQUIP) method for accurate absolute quantification of protein PTM isoforms.
  • To address the challenges in absolute quantification, including protein fractionation, enrichment, and digestion yield.
  • To provide a framework for calculating absolute protein PTM isoform amounts from protein and PTM occupancy data.

Main Methods:

  • Utilizes liquid chromatography-tandem mass spectrometry (LC-MS/MS) with stable isotope-coded peptide standards.
  • Incorporates correction for impacts of protein fractionation, enrichment, and proteolytic digestion yield.
  • Employs affinity purification of tagged recombinant proteins and stable isotope-labeled peptide standards.

Main Results:

  • The AQUIP method determines absolute protein PTM isoform amounts by combining absolute protein quantity and PTM occupancy.
  • Absolute protein quantity is inferred from PTM-site-independent peptides and their yields.
  • PTM occupancy is measured by quantifying PTM and non-PTM peptides from purified samples.

Conclusions:

  • The AQUIP method enables accurate absolute quantification of protein PTM isoforms.
  • This approach accounts for critical experimental variables affecting quantitation accuracy.
  • The described methodology enhances the biological significance of quantitative proteomics studies.