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

Yassene Mohammed, Jingxi Pan, Suping Zhang, Jun Han, Christoph H. Borchers

Research output: Contribution to journalArticlepeer-review

15 Citations (Scopus)


Purpose: Targeted proteomics using MRM with stable-isotope-labeled internal-standard (SIS) peptides is the current method of choice for protein quantitation in complex biological matrices. Better quantitation can be achieved with the internal standard-addition method, where successive increments of synthesized natural form (NAT) of the endogenous analyte are added to each sample, a response curve is generated, and the endogenous concentration is determined at the x-intercept. Internal NAT-addition, however, requires multiple analyses of each sample, resulting in increased sample consumption and analysis time. Experimental design: To compare the following three methods, an MRM assay for 34 high-to-moderate abundance human plasma proteins is used: classical internal SIS-addition, internal NAT-addition, and external NAT-addition—generated in buffer using NAT and SIS peptides. Using endogenous-free chicken plasma, the accuracy is also evaluated. Results: The internal NAT-addition outperforms the other two in precision and accuracy. However, the curves derived by internal vs. external NAT-addition differ by only ≈3.8% in slope, providing comparable accuracies and precision with good CV values. Conclusions and clinical relevance: While the internal NAT-addition method may be “ideal”, this new external NAT-addition can be used to determine the concentration of high-to-moderate abundance endogenous plasma proteins, providing a robust and cost-effective alternative for clinical analyses or other high-throughput applications.

Original languageEnglish
Article number1600180
JournalProteomics - Clinical Applications
Issue number2
Publication statusPublished - Mar 2018
Externally publishedYes


  • ExSTA
  • external standard addition
  • Multiple Reaction Monitoring (MRM)
  • quantitative proteomics
  • standard addition
  • standard curve


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