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Targeted absolute quantitative proteomics with SILAC internal standards and unlabeled full‐length protein calibrators (TAQSI)
Author(s) -
Wang Xinwen,
Liang Yan,
Liu Li,
Shi Jian,
Zhu HaoJie
Publication year - 2016
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.7482
Subject(s) - stable isotope labeling by amino acids in cell culture , quantitative proteomics , proteomics , chemistry , chromatography , label free quantification , computational biology , bradford protein assay , proteome , biochemistry , biology , gene
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. Copyright © 2016 John Wiley & Sons, Ltd.