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Proteomics Clin Appl. 2018 Mar;12(2). doi: 10.1002/prca.201600180. Epub 2017 Oct 25.

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

Author information

1
University of Victoria - Genome British Columbia Proteomics Centre, Victoria, Canada.
2
Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands.
3
MRM Proteomics Inc., Victoria, British Columbia, Canada.
4
University of Victoria, Department of Biochemistry and Microbiology, Victoria, BC, Canada.
5
Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, Quebec, Canada.
6
Proteomics Centre, Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, Quebec, Canada.

Abstract

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.

KEYWORDS:

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

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