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. 2018 Mar;12(2):1600180.
doi: 10.1002/prca.201600180. Epub 2017 Oct 25.

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

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Free PMC article

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

Yassene Mohammed et al. Proteomics Clin Appl. .
Free PMC article

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.

Figures

Figure 1
Figure 1
The three methods for generating standard curves. The method shown in panel A—the internal SIS‐addition method—illustrates the common method for generating the standard curve in sample, which uses a SIS peptide mixture spiked in at several concentration levels. The calibration curve is generated by plotting the SIS/END peak area ratios as a function of SIS peptide concentration. The method shown in panel B shows the internal NAT‐addition method where a concentration‐balanced SIS peptide mixture (SISc) is spiked into the sample at a single concentration level and the NAT is added at several concentration levels. The standard curve is generated by plotting the relative response (i.e., R(NAT + END)/RSISc), as a function of the spiked‐in NAT peptide concentration. The concentration of the END peptide is estimated by extrapolating the standard curve and determining the x‐intercept, of which the END concentration is the absolute value. Panel C shows the external NAT‐addition method—ExSTA. In this method, the SISc peptide mixture is spiked into buffer at a single concentration level, and varying levels of NAT are spiked into the mixture. The peak areas of the NAT and SISc peptides are used (the END peptide is not present). After generating the calibration curve in buffer, the END peptide concentration in a sample is estimated from a single point measurement of a sample, to which only a single concentration level of SISc peptide has been added. The yellow lines in all three panels represent the confidence interval associated with the standard curve.
Figure 2
Figure 2
A box‐and‐whisker plot of the R 2 values for the three methods used to generate the standard curves. One outlier in the external NAT‐addition method—ExSTA, originated from antithrombin‐III peptide DDLYVSDAFHK, had an R 2 value of 0.53. The spread of the boxplot corresponds to the variability in the method.
Figure 3
Figure 3
Scatter plots showing the correlation between the concentrations determined by the internal SIS‐addition method and the internal NAT‐addition method. The four plots show four different plotting ranges and the corresponding measured peptide concentration in these ranges, these are 0 to 5e5 fmol/μL for the full range, 1000 to 5000 fmol/μL, 100 to 1000 fmol/μL, and 0 to 100 fmol/μL.
Figure 4
Figure 4
Scatter plots showing the correlation between the concentrations determined by the internal and external NAT‐addition methods, both using the same concentration‐balanced SIS and NAT peptides. The four plots show four different plotting ranges and the corresponding measured peptide concentration in these ranges, these are 0 to 2e5 fmol/μL for the full range, 1000 to 5000 fmol/μL, 100 to 1000 fmol/μL, and 0 to 100 fmol/μL.
Figure 5
Figure 5
Boxplot of the percent recovery in each of the three methods, internal NAT addition, external NAT addition—ExSTA, and internal SIS‐addition methods. Extreme values appearing as outliers are marked with the name of the corresponding protein.

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