Quantitative performance of internal standard platforms for absolute protein quantification using multiple reaction monitoring-mass spectrometry.
Anal Chem
; 87(8): 4429-35, 2015 Apr 21.
Article
em En
| MEDLINE
| ID: mdl-25812027
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
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Interferon gama
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Fator de Necrose Tumoral alfa
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Interleucina-1beta
Limite:
Humans
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2015
Tipo de documento:
Article
País de afiliação:
Estados Unidos