Absolute Protein Quantification by LC-MS/MS: Comparison of Peptide Isotope Ratio and Protein Calibration Curve Back-Calculation, presented at the AAPS National Biotechnology Conference, May 20-22, 2013 in San Diego, CA.

Purpose: Protein quantification in biological samples using LC-MS/MS is predominantly done via surrogate peptides. Two quantification methods of the target peptides are commonly used: peptide isotope
ratio, where the ratio between SIL-peptide response and the native peptide response is used to determine
the amount of protein, and generation of a protein standard curve over a range of concentrations and
back-calculation of analyte concentration from the equation of the line derived from weighted linear
regression analysis. We present a study to directly compare each approach for absolute quantitation of
proteins by LC-MS/MS.
Methods: UGT isozymes were quantified in individual donor liver microsomes via both quantification
methods described. Liver microsomes were reduced, alkylated, and digested with trypsin overnight.
Stable isotope-labeled peptides were added to each sample as internal standards. Traditional calibration
standards were prepared by adding known amounts of recombinant protein preparations into rat liver
microsomes. Multiple unique tryptic peptides for each isozyme were monitored by SRM or MS3 using an
AB Sciex mass spectrometer interfaced to an eksigent microUPLC system. The values for absolute
amount of protein were determined using the isotope ratio approach or the calibration curve.
Preliminary Data: Assays for the quantification of six UGT enzyme isoforms by LC-MS/MS were
developed and tested in individual human liver microsomes. Replicate calibration curves (n=3-5) exhibited
appropriate accuracy (20% of nominal) and precision (20% CV) across the protein calibration curve. In all
cases, the use of the peptide isotope ratio approach yielded lower concentrations than the protein
calibration curve approach. The difference in the calculated concentrations between the 2 methods of
protein quantification ranged from 20 to 90% difference. This observation can be explained by considering
that protein digestion is not 100% efficient and that the assumption inherent in the isotope ratio approach,
namely, that each molecule of protein yields one molecule of peptide, is not always a valid assumption.
However, both the protein calibration curve and the peptide isotope ratio approaches generate equivalent
relative concentration values.
Conclusion: When needed, absolute quantification is appropriately done using a protein calibration curve, rather than peptide isotope ratio.


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