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; 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 were
acquired by data-dependent acquisition (DDA) and SWATH-MSALL 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 (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, p < 0.001). Protein-to-activity
correlation was moderate (r > 0.400–0.600, p < 0.001) for CYP1A2, CYP2A6, CYP2B6, CYP2C9, and CYP2E1
and significant but poor (r < 0.400, p < 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.