期刊
JOURNAL OF PROTEOME RESEARCH
卷 16, 期 7, 页码 2527-2536出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.7b00094
关键词
peptide quantitation; MRM; stable isotope-labeled peptides; surrogate standard; surrogate matrix; peptide calibration; method development; peptide assay validation; peptide isotopologues
资金
- Genome Canada
- Genome British Columbia [204PRO, 214PRO]
- Leading Edge Endowment Fund (LEEF)
- Segal McGill Chair in Molecular Oncology at McGill University (Montreal, Quebec, Canada)
- Warren Y. Soper Charitable Trust
- Alvin Segal Family Foundation
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.
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