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ExSTA: External Standard Addition Method for Accurate High-Throughput Quantitation in Targeted Proteomics

Yassene Mohammed1,2, Jingxi Pan1, Suping Zhang3

  • 1University of Victoria - Genome British Columbia Proteomics Centre, Victoria, Canada.

Proteomics. Clinical Applications
|September 13, 2017
PubMed
Summary

External NAT-addition offers a robust, cost-effective alternative for quantifying plasma proteins, matching the precision and accuracy of internal methods while reducing sample analysis time and consumption.

Keywords:
ExSTAMultiple Reaction Monitoring (MRM)external standard additionquantitative proteomicsstandard additionstandard curve

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Targeted proteomics with MRM and SIS peptides is standard for protein quantitation.
  • Internal standard-addition methods improve quantitation but increase analysis time and sample use.

Purpose of the Study:

  • To compare classical internal SIS-addition, internal NAT-addition, and external NAT-addition for protein quantitation.
  • To evaluate accuracy using endogenous-free chicken plasma.

Main Methods:

  • MRM assay for 34 human plasma proteins.
  • Comparison of internal SIS-addition, internal NAT-addition, and external NAT-addition.
  • Accuracy assessment using chicken plasma.

Main Results:

  • Internal NAT-addition showed superior precision and accuracy.
  • External NAT-addition curves differed minimally (≈3.8% slope) from internal NAT-addition.
  • Both NAT-addition methods provided comparable accuracy and precision.

Conclusions:

  • External NAT-addition is a viable, cost-effective alternative to internal methods for high-throughput protein quantitation.
  • This method is suitable for clinical analyses and other applications.
  • It offers a practical solution for complex biological matrices.