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Characterization of Stable Isotope Labeled Human Protein Standards for Quantitative Mass Spectrometry Based Assays

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

Abstract

The application of mass spectrometry for protein quantitation has been increasing steadily over the last decade, particularly for applications in which the availability of suitable antibody reagents is limited. Stable-isotope labeled (SIL) synthetic peptides are typically used as internal standards for the quantitation of proteins by mass spectrometry. The method is often extended with the use of anti-peptide antibodies to capture low abundance peptides of interest, termed SISCAPA. The accuracy of absolute quantitation with such peptide-based approaches is subject to error associated with protein fractionation, enrichment, and proteolysis steps. A more ideal alternative strategy involves the use of SIL proteins that can be introduced early in the analytical workflow as true internal standards. In this study, we describe the characterization of stable-isotope labeled full-length proteins (SILu™Prot APOA1, SILu™Prot IL6RA, SILu™Prot HPT, SILu™Prot ANT3) generated by baculovirus-directed expression in human HEK293 cells and demonstrate their use as internal standards for MS-based protein quantitation.

Results

hpt

Figure 1. SDS-PAGE of “heavy” Apo-A1, IL6RA  and HPT incorporating 13C615N4 Arg / 13C615N2 Lys (upper panel). The sequence coverage (red text observed) for APO-A1 tryptic digest was found to be 91.2% (lower panel).
 

sigma-r-d-batch-commercial

Figure 2. Incorporation of 13C615N2 labeled lysine in DYVSQFEGSALGK (APO-A1) from the Sigma R&D batch and a commercially sourced “heavy” Apo-A1.
 

peptide-sequence

Figure 3.  Incorporation of stable labeled isotope for selected representative peptides from “heavy” Apo-A1 from the Sigma R&D batch and a commercially sourced “heavy” Apo-A1.
 

mrm-traces

Figure 4.  MRM traces of three APO-A1 peptides with three transitions each.
 

calibration-curves

Figure 5. Calibration curves of three ANT3 peptides obtained by spiking human serum samples with “heavy” ANT3 protein at levels from 2 µg/mL to 500 µg/mL.
 

multiple-peptides

Figure 6. Multiple peptides derived from “heavy” ANT3 were utilized to determine the endogenous level of ANT3 present in a human serum sample.
 

Summary

  • Stable isotope labeled “heavy” full-length proteins have been produced in HEK293 cells with purity and isotopic incorporation >95%
  • MRM transitions were optimized for three surrogate peptides derived from each “heavy” protein
  • Calibration studies yielded reproducible, linear curves from 2 µg/mL to 500 µg/mL in human serum matrix without enrichment or depletion
  • Good agreement between multiple peptides derived from the same target protein was observed for the quantitation of endogenous serum proteins and the results were consistent with literature values
  • SIL proteins offer a more suitable choice for use as an internal standard in quantitative mass spectrometric based protein assays

Reference

  1. Anderson L. Candidate-based proteomics in the search for biomarkers of cardiovascular disease. The Journal of physiology. 2005; 1:23–60