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A multiplex and label-free relative quantification approach for studying protein abundance of drug metabolizing enzymes in human liver microsomes using SWATH-MS Rohitash Jamwal, Benjamin J. Barlock, Sravani Adusumalli, Ken Ogasawara, Brigitte L. Simons, and Fatemeh Akhlaghi J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00505 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 26, 2017
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Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Journal of Proteome Research
A multiplex and label-free relative quantification approach for studying protein abundance of drug metabolizing enzymes in human liver microsomes using SWATH-MS
Rohitash Jamwal1, Benjamin J. Barlock1, Sravani Adusumalli1, Ken Ogasawara1, Brigitte L. Simons2, Fatemeh Akhlaghi1*
1
Clinical Pharmacokinetics Research Laboratory, Department of Biomedical and Pharmaceutical
Sciences, University of Rhode Island, Kingston, 02881, RI, USA 2
SCIEX, 71 Four Valley Dr., Concord, Ontario, L4K4V8, Canada
Author for correspondence: *Fatemeh Akhlaghi, Ph.D., PharmD Clinical Pharmacokinetics Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, 495A College of Pharmacy, 7 Greenhouse Road, Kingston, RI 02881, United States. Email address:
[email protected] Keywords: Proteomics, SWATH-MS, label-free quantification, human liver, cytochrome-P450, CYP3A4
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Journal of Proteome Research
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List of nonstandard abbreviations BHT: Butylated hydroxytoluene; CYP: Cytochrome P450; DMEs: Drug metabolizing enzymes; DDA: Data dependent analysis; FDR: False discovery rate; HLM: Human liver microsomes; LFQ: Label-free quantification; MRM: Multiple reaction monitoring; PCT: Pressure cycling technology; SRM: Single-reaction monitoring; SWATH-MS: Sequential windowed acquisition of all theoretical fragment ion mass spectra; TPCK: Tosyl phenylalanyl chloromethyl ketone; UGT: UDP-glucuronosyltransferase; UHPLC: Ultra high performance liquid chromatography.
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Journal of Proteome Research
ABSTRACT We describe a sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) based method for label-free, simultaneous, relative quantification of drug metabolism enzymes in human liver microsomes (HLM; n=78). In-solution tryptic digestion was aided by a pressure cycling method which allowed a 90-min incubation time, a significant reduction over classical protocols (12-18 h). Digested peptides were separated on an Acquity UHPLC Peptide BEH C18 column using a 60-min gradient method at a flow rate of 0.100 mL/min. The quadrupole-time-of-flight mass spectrometer (ESI-QTOFMS) was operated in positive electrospray ionization mode and data was acquired by Data-Dependent Acquisition (DDA) and SWATH-MSALL mode. A pooled HLM sample was used as quality control to evaluate variability in digestion and quantification among different batches, and inter-batch %CV for various proteins was between 3.1-7.8%. Spectral library generated from the DDA data identified 1,855 distinct proteins and 25,601 distinct peptides at a 1% global false discovery rate (FDR). SWATH data was queried and analyzed for 10 major cytochrome P450 (CYP) enzymes using Skyline, a targeted data extraction software. Further, correlation analysis was performed between functional activity, protein and mRNA expression for ten CYP enzymes. Pearson correlation coefficient (r) between protein and activity for CYPs ranged from 0.314 (CYP2C19) to 0.767 (CYP2A6). A strong correlation was found between CYP3A4 and CYP3A5 abundance and activity determined using midazolam and testosterone (r>0.600, p0.500, p
