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A Comparison of Peptides to Whole-Protein Stable Isotope Labeled Internal Standards in Quantitative Protein MS Workflows

By: James J Walters, Pegah Jalili, Kevin Ray, Sigma-Aldrich, 2909 Laclede Ave, St. Louis, MO, 63103

Objective

Characterize a stable isotope labeled human erythropoietin (SILu™Prot EPO) and compare it’s use as an internal standard in an immunoaffinity enrichment experiment to the SIL peptide approach.

Overview

LC-MS/MS methods employing the sensitivity of multiple reaction monitoring (MRM) for quantification of proteins has typically utilized stable isotope labeled peptides (SIL-Pep), commonly called AQUA peptides. While good reproducibility and relative quantitation can be achieved with such peptide-based approaches, the accuracy of absolute quantitation is subject to error associated with protein fractionation, enrichment, and proteolysis steps that generally occur prior to introduction of the stable-labeled peptide. A more ideal alternative strategy involves the use of a stable labeled full length protein internal standard that can be introduced early in the analytical workflow, overcoming processing variability. In this study, we compared the performance of SIL peptides to whole-molecule SIL protein as the internal standard in a quantitative assay for Erythropoietin (EPO) in plasma. The results demonstrate that the performance of whole-protein SIL-EPO is better than that of SIL peptides in this application, likely owing to compensation for inefficiencies in the immunoaffinity enrichment and digestion steps of the assay.

Methods

methods

Results

purity-peptide

Figure 1. SIL-EPO purity was visualized by 1-D PAGE. The excised band underwent enzymatic digestion followed by LC-MS/MS analysis of the resulting tryptic peptides on a Thermo LTQ-FT. Resulting data was database searched, >75% sequence coverage was observed. Highlighted in the sequence are the surrogate peptides used for quantitative analysis.
 

sil-incorporation

Figure 2. Incorporation of stable labeled isotopes for surrogate peptides from the SIL-EPO protein are shown. Data was generated by LC-MS/MS. Incorporation was demonstrated to be greater than 98%.
 

lc-ms

Figure 3. Extracted ion chromatogram (XIC) of three EPO peptides from a digested IA enriched sample containing 50 ng/mL SIL-EPO, 100 ng/mL human EPO standard in a beagle plasma matrix. On-plate tryptic digestion was followed by dry down and reconstitution in 0.1% formic acid.
 

protein-is

Figure 4. Standard curves for Protein and Peptide IS (Internal Standard) experiments. Data points represent an average of three process replicates.
 

accuracy-peptide-protein

Figure 5. Comparison of the accuracy of data points obtained using either Peptide or Protein IS.

Summary

The performance of SIL peptides to whole-molecule SIL protein as the internal standard in a quantitative assay for Erythropoietin (EPO) in plasma was compared.

Whole-protein SIL-EPO provided more accurate quantitative data than that of SIL peptides based on linearity and accuracy.