Use of SWATH-MS to relatively quantify the expression of hepatic drug metabolizing enzymes in human liver microsomes using a label-free approach Rohitash Jamwal, Benjamin J. Barlock, Sravani Adusumalli, Ken Ogasawara, Brigitte L. Simons, Fatemeh Akhlaghi 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; male=41, female=37). SWATH-MS exemplify a powerful technique, which addresses the limitations of the shotgun and targeted proteomics to provide a permanent digital repository of all peptides present in a sample. This method can serve as a valuable discovery tool as well as a post hoc tool for studying new hypothesis and ideas without the need to re-acquire data. In-solution tryptic digestion was aided by a pressure cycling method which allowed a short 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-MS mode. A pooled HLM sample was used to evaluate variability in digestion and quantification among different batches, and inter-batch %CV for proteins of interest was <17%. An in-house generated spectral library was created and 1,855 distinct proteins and 25,601 peptides at a global false discovery rate (FDR) of 1% were identified. SWATH-MS data was queried and analyzed for major cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) isoforms (23 and 13 respectively) using Skyline software. The utility of the current method was evaluated by performing a three-way correlation analysis between functional activities, protein and mRNA expression for ten CYP and four UGT enzymes. Other drug metabolizing enzymes such as carboxylesterases (CES) and flavin-containing monooxygenases (FMO) were quantified in the same way using Skyline software but no functional activity assays or mRNA expressions where measured. Pearson correlation coefficient (r) for CYPs and UGTs protein and activity ranged from 0.170 (CYP2B6) to 0.733 (CYP2E1). A high correlation was found between CYP3A4/3A5 abundance, and activity (determined using midazolam and testosterone). A strong protein-to-activity correlation (r>0.500, p<0.01) was also observed for CYP2A6, CYP1A2, CYP2C9, CYP2E1 and UGT1A4. The correlation for CYP2C8, CYP2C19, UGT1A1 and UGT2B7 was significant and moderately strong (r~0.400, p<0.05). The relationship between CYP2B6 and UGT1A6 protein level and activity was not significant (p=0.168 and 0.206, respectively). Overall, the findings suggest the suitability of SWATH-MS based method as a valuable and relatively fast analysis technique for relative quantification of proteins in biological specimen in a multiplex fashion.