Quantitative performance of internal standard platforms for absolute protein quantification using multiple reaction monitoring-mass spectrometry.

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TitleQuantitative performance of internal standard platforms for absolute protein quantification using multiple reaction monitoring-mass spectrometry.
Publication TypeJournal Article
Year of Publication2015
AuthorsScott, KBauer, Turko, IV, Phinney, KW
JournalAnal Chem
Volume87
Issue8
Pagination4429-35
Date Published2015 Apr 21
ISSN1520-6882
Abstract<p>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.</p>
DOI10.1021/acs.analchem.5b00331
Alternate JournalAnal. Chem.
PubMed ID25812027