Multiplexed MRM-Based Protein Quantitation Using Two Different Stable Isotope-Labeled Peptide Isotopologues for Calibration.
J Proteome Res
; 16(7): 2527-2536, 2017 07 07.
Article
em En
| MEDLINE
| ID: mdl-28516774
ABSTRACT
When quantifying endogenous plasma proteins for fundamental and biomedical research - as well as for clinical applications - precise, reproducible, and robust assays are required. Targeted detection of peptides in a bottom-up strategy is the most common and precise mass spectrometry-based quantitation approach when combined with the use of stable isotope-labeled peptides. However, when measuring protein in plasma, the unknown endogenous levels prevent the implementation of the best calibration strategies, since no blank matrix is available. Consequently, several alternative calibration strategies are employed by different laboratories. In this study, these methods were compared to a new approach using two different stable isotope-labeled standard (SIS) peptide isotopologues for each endogenous peptide to be quantified, enabling an external calibration curve as well as the quality control samples to be prepared in pooled human plasma without interference from endogenous peptides. This strategy improves the analytical performance of the assay and enables the accuracy of the assay to be monitored, which can also facilitate method development and validation.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
/
Espectrometria de Massas
/
Bioensaio
/
Proteínas Sanguíneas
/
Cromatografia Líquida
/
Proteômica
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article