Multiplex and Label-Free Relative Quantification Approach for Studying Protein Abundance of Drug Metabolizing Enzymes in Human Liver Microsomes Using SWATH-MS

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; <i>n</i> = 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 were acquired by data-dependent acquisition (DDA) and SWATH-MS^ALL mode. A pooled HLM sample was used as a quality control to evaluate variability in digestion and quantification among different batches, and inter-batch %CV for various proteins was between 3.1 and 7.8%. Spectral library generated from the DDA data identified 1855 distinct proteins and 25 681 distinct peptides at a 1% global false discovery rate (FDR). SWATH data were 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 (<i>r</i>) 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 (<i>r</i> > 0.600, <i>p</i> < 0.001). Protein-to-activity correlation was moderate (<i>r</i> > 0.400–0.600, <i>p</i> < 0.001) for CYP1A2, CYP2A6, CYP2B6, CYP2C9, and CYP2E1 and significant but poor (<i>r</i> < 0.400, <i>p</i> < 0.05) for CYP2C8, CYP2C19, and CYP2D6. The findings suggest the suitability of SWATH-MS based method as a valuable and relatively fast analytical technique for relative quantification of proteins in complex biological samples. We also show that protein abundance is a better surrogate than mRNA to predict the activity of CYP activity