Ultrasensitive microflow‐UPLC‐MS/MS method for quantifying drug metabolizing enzymes and transporters in tissue S9 fractions applicable to small total protein amount and biopsy samples

Abstract

Quantitative proteomics is a powerful technique that provides important data for in vitro to in vivo extrapolation (IVIVE) and predicts interindividual variability in drug disposition.However, the conventional quantitative proteomics methods often require high initial total protein and enriched samples (e.g., microsomes and pure membrane fractions). The goal of this study was i) to develop a microflow based LC‐MS/MS method for quantification of clinically relevant drug metabolizing enzymes (DMEs) and transporters using small total protein (400 ng), and ii) to quantify these proteins in human liver and intestine S9 samples.Targeted quantitation of DMEs and transporters was performed on trypsin digested pooled liver (N=200) and pooled intestinal (N=15) S9 fractions (Xenotech, Kansas city). The digestion was performed using an optimized in house protocol. A total of 400 ng protein (1 μl injection volume) was injected through a trap‐column followed by peptide separation in a microflow column (BEH C18; 130 A°,1.7μm, 150 μm*50 mm) using M‐class UPLC system and detection by Xevo TQ‐XS MS instrument supported by ionkey interface. An optimized gradient elution was used with 3 μl/min flow rate. Data were acquired in scheduled MRM mode and the data analysis was performed using Skyline 19.1 software.72 DMEs and transporters were quantifiable using 400 ng of total protein injection. A total of 12 CYPs, 12 UGTs, 33 nonCYPs/nonUGTs (e.g., SULTs, GSTs, ADHs, ALDHs, etc.) and 16 transporters were detected. Out of the total detected proteins, 53 were in both the liver and intestine, 17 were only detected in the liver, and 2 were only quantifiable in the intestine. CYP2C8, CYP2B6, CYP2D6, CYP2A6, CYP2E1, UGT1A9, UGT1A4, UGT2B4, UGT2B15, SULT1E1, PON3, FMO3, AOX1, ADH1A, ADH1B, OATP1B1 and OATP1B3 were exclusively detected in the liver whereas UGT1A10 and UGT2A1 were only seen in the intestine. ALDH1A1, GSTT1, SULT4A1, SLC22A3, CYP3A4, ALDH1A3, SULT2A1, CYP1A1, SLC22A2, MATE1, ADH1C, SULT1C, POR, ABCC2 were within 0.75–1.5‐fold in the liver and intestine S9 fractions. CES1, PON1, UGT1A6, CYP2C9 and UGT2B7 were significantly higher in the liver as compared to intestine, and SULT1B1, CES2, UGT2B17, FMO1 and UGT1A8 were amongst the top differentially expressed proteins in the intestine. We were able to quantify low abundant proteins such as BCRP, CYP1A1 and UGT1A8, which are generally difficult to detect by conventional LC‐MS/MS methods. In summary, the microflow‐LC‐MS/MS is an ultrasensitive approach for quantifying DMEs and transporters in crude tissue preparations such as homogenate and S9 fractions. Since microsomes and pure membrane preparations require recovery factors that add batch‐to‐batch variability, quantification of proteins in homogenate and S9 fractions using the ultrasensitive method would provide accurate scaling factors for IVIVE. This microflow‐LC/MS/MS method can be applied to small biopsy samples (e.g., tissue weight ~2.5 mg) thus providing broad applications in predicting interindividual variability (e.g., effect of diseases) in drug disposition using patient samples.Support or Funding InformationDepartment of Pharmaceutical Sciences, Washington State UniversityNumber of proteins quantified in intestine and liver S9 fractions.Figure 1