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ABSTRACT: Rationale
Liquid Chromatography/Mass Spectrometry (LC/MS)-based proteomics for absolute protein quantification has been increasingly utilized in both basic and clinical research. There is a great need to overcome some major hurdles of current absolute protein quantification methods, such as significant inter-assay variability and the high cost associated with the preparation of purified stable-isotope-labeled peptide/protein standards.Methods
We developed a novel targeted absolute protein quantification method, named TAQSI, utilizing full-length isotope-labeled protein internal standards generated from SILAC (stable isotope labeling by amino acid in cell culture) and unlabeled full-length protein calibrators. This approach was applied to absolute quantification of carboxylesterase 1 (CES1), the primary human hepatic hydrolase, in a large set of human liver samples. Absolute CES1 quantities were derived from the standard calibration curves established from unlabeled CES1 protein calibrators and the isotope-labeled CES1 internal standards obtained from SILAC HepG2 cells.Results
The TAQSI assay was found to be accurate, precise, reproducible, and cost-effective. Importantly, protein quantification was not affected by various protein extraction and digestion protocols, and measurement errors associated with nonsynonymous variants can be readily identified and avoided. Furthermore, the TAQSI approach significantly simplifies the procedure of identifying the best performance surrogate peptides.Conclusions
The TAQSI assay can be widely used for targeted absolute protein quantification in various biomedical research and clinical practice settings.
SUBMITTER: Wang X
PROVIDER: S-EPMC6301059 | biostudies-literature | 2016 Mar
REPOSITORIES: biostudies-literature
Wang Xinwen X Liang Yan Y Liu Li L Shi Jian J Zhu Hao-Jie HJ
Rapid communications in mass spectrometry : RCM 20160301 5
<h4>Rationale</h4>Liquid Chromatography/Mass Spectrometry (LC/MS)-based proteomics for absolute protein quantification has been increasingly utilized in both basic and clinical research. There is a great need to overcome some major hurdles of current absolute protein quantification methods, such as significant inter-assay variability and the high cost associated with the preparation of purified stable-isotope-labeled peptide/protein standards.<h4>Methods</h4>We developed a novel targeted absolut ...[more]