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Multiple-Enzyme-Digestion Strategy Improves Accuracy and Sensitivity of Label- and Standard-Free Absolute Quantification to a Level That Is Achievable by Analysis with Stable Isotope-Labeled Standard Spiking
Max Planck Inst Biochem, Dept Prote & Signal Transduct, Biochem Prote Grp, Klopferspitz 18, D-82152 Martinsried, Germany.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. AstraZeneca, Cardiovasc Renal & Metab, Innovat Med & Early Dev Biotech Unit, Gothenburg, Sweden.ORCID iD: 0000-0002-2810-7518
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
2019 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 18, no 1, p. 217-224Article in journal (Refereed) Published
Abstract [en]

Quantification of individual proteins is an essential task in understanding biological processes. For example, determination of concentrations of proteins transporting and metabolizing xenobiotics is a prerequisite for drug disposition predictions in humans based on in vitro data. So far, this task has frequently been accomplished by targeted proteomics. This type of analyses requires preparation of stable isotope labeled standards for each protein of interest. The selection of appropriate standard peptides is usually tedious and the number of proteins that can be studied in a single experiment by these approaches is limited. In addition, incomplete digestion of proteins often affects the accuracy of the quantification. To circumvent these constrains in proteomic protein quantification, label- and standard-free approaches, such as "total protein approach" (TPA) have been proposed. Here we directly compare an approach using stable isotope labeled (SIL) standards and TPA for quantification of transporters and enzymes in human liver samples within the same LC-MS/MS runs. We show that TPA is a convenient alternative to SIL-based methods. Optimization of the sample preparation beyond commonly used single tryptic digestion, by adding consecutive cleavage steps, improves accuracy and reproducibility of the TPA method to a level, which is achievable by analysis using stable isotope-labeled standard spiking.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC , 2019. Vol. 18, no 1, p. 217-224
Keywords [en]
FASP, MED FASP, total protein approach (TPA), stable isotope labeling, quantitative analysis, drug transporter, drug metabolizing enzymes, ADME
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-375231DOI: 10.1021/acs.jproteome.8b00549ISI: 000455285900019PubMedID: 30336047OAI: oai:DiVA.org:uu-375231DiVA, id: diva2:1283655
Funder
Swedish Research Council, 1951Swedish Research Council, 5715Available from: 2019-01-29 Created: 2019-01-29 Last updated: 2019-07-23Bibliographically approved

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