Protein quantification using a cleavable reporter peptide

Elodie Duriez, Stephane Trevisiol, Bruno Domon*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

7 Citations (Scopus)

Abstract

Peptide and protein quantification based on isotope dilution and mass spectrometry analysis are widely employed for the measurement of biomarkers and in system biology applications. The accuracy and reliability of such quantitative assays depend on the quality of the stable-isotope labeled standards. Although the quantification using stable-isotope labeled peptides is precise, the accuracy of the results can be severely biased by the purity of the internal standards, their stability and formulation, and the determination of their concentration. Here we describe a rapid and cost-efficient method to recalibrate stable isotope labeled peptides in a single LC-MS analysis. The method is based on the equimolar release of a protein reference peptide (used as surrogate for the protein of interest) and a universal reporter peptide during the trypsinization of a concatenated polypeptide standard. The quality and accuracy of data generated with such concatenated polypeptide standards are highlighted by the quantification of two clinically important proteins in urine samples and compared with results obtained with conventional stable isotope labeled reference peptides. Furthermore, the application of the UCRP standards in complex samples is described.

Original languageEnglish
Pages (from-to)728-737
Number of pages10
JournalJournal of Proteome Research
Volume14
Issue number2
DOIs
Publication statusPublished - 6 Feb 2015

Keywords

  • calibration
  • cleavable reporter peptide
  • mass spectrometry
  • peptide/protein quantification
  • stable isotope labeled peptides
  • standard recalibration
  • targeted proteomics

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