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Quantitative performance of internal standard platforms for absolute protein quantification using MRM-MS



Kerry M. Bauer, Illarion V. Turko, Karen W. Phinney


Isotope dilution mass spectrometry involves the addition of known quantities of isotope-labeled standards, that mimic native molecules, to biological samples. We evaluated three conventional internal standard platforms (synthetic peptides, QconCAT constructs, and PSAQ proteins) for quantitative accuracy, precision, and inherent advantages and limitations. Internal standards for the absolute quantification of three human cytokine proteins (interferon gamma, interleukin-1 beta, and tumor necrosis factor) were designed and verified. Multiple reaction monitoring assays, calibration curve construction, and regression analysis were used to assess quantitative performance of the internal standard platforms. We also investigated a strategy for methodological improvement to current platforms using natural flanking sequences. Data analysis revealed that full length protein standards have the broadest quantitative reliability with accuracy being peptide-dependent for QconCATs and synthetic peptides. Natural flanking sequences greatly improved the quantitative performance of both QconCAT and synthetic peptide standards.
Analytical Chemistry


IDMS, isotope dilution mass spectrometry, MRM, multiple reaction monitoring, PSAQ, protein standard absolution quantification, QconCAT, quantification concatamer


Bauer, K. , Turko, I. and Phinney, K. (2015), Quantitative performance of internal standard platforms for absolute protein quantification using MRM-MS, Analytical Chemistry, [online], (Accessed April 13, 2024)
Created March 26, 2015, Updated November 10, 2018