title: Targeted absolute quantitative proteomics with SILAC internal standards and unlabeled full-length protein calibrators (TAQSI).
abstract: BACKGROUND
  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.
authors: Department of Clinical Pharmacy, University of Michigan, Ann Arbor, MI, 48109-1065, USA.
authors: Xinwen
authors: X
authors: Wang
authors: Department of Clinical Pharmacy, University of Michigan, Ann Arbor, MI, 48109-1065, USA.|The Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, China.
authors: Yan
authors: Y
authors: Liang
authors: Department of Clinical Pharmacy, University of Michigan, Ann Arbor, MI, 48109-1065, USA.|The Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, China.
authors: Li
authors: L
authors: Liu
authors: Department of Clinical Pharmacy, University of Michigan, Ann Arbor, MI, 48109-1065, USA.
authors: Jian
authors: J
authors: Shi
authors: Department of Clinical Pharmacy, University of Michigan, Ann Arbor, MI, 48109-1065, USA.
authors: Hao-Jie
authors: HJ
authors: Zhu
mesh_terms: D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases / Q000032:analysis*; D002853:Chromatography, Liquid / Q000379:methods; D056945:Hep G2 Cells; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D008099:Liver / Q000737:chemistry / Q000201:enzymology*; D010455:Peptides / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010455:Peptides; D002265:Carboxylic Ester Hydrolases; C474609:CES1 protein, human