title: Quantitative performance of internal standard platforms for absolute protein quantification using multiple reaction monitoring-mass spectrometry.
abstract: Stable-isotope-labeling mass spectrometry involves the addition of known quantities of stable-isotope labeled standards, which mimic native molecules, to biological samples. We evaluated three conventional internal standard platforms (synthetic peptides, QconCAT constructs, and recombinant 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 alpha) 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.
authors: †Biomolecular Measurement Division, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899, United States.|‡Institute for Bioscience and Biotechnology Research, 9600 Gudelsky Drive, Rockville, Maryland 20850, United States.
authors: Kerry Bauer
authors: KB
authors: Scott
authors: †Biomolecular Measurement Division, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899, United States.|‡Institute for Bioscience and Biotechnology Research, 9600 Gudelsky Drive, Rockville, Maryland 20850, United States.
authors: Illarion V
authors: IV
authors: Turko
authors: †Biomolecular Measurement Division, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899, United States.
authors: Karen W
authors: KW
authors: Phinney
mesh_terms: D006801:Humans; D007371:Interferon-gamma / Q000032:analysis*; D053583:Interleukin-1beta / Q000032:analysis*; D013058:Mass Spectrometry; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*
chemical_list: D053583:Interleukin-1beta; D010455:Peptides; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma