Article Figures & Data
Figures
- Fig. 1.
Representative CYP3A4 stability data for human HepatoPac cocultures through the time period used for P450 degradation rate determinations. Data are mean from n = 3 replicates with error bars indicating S.D. mRNA expressions are normalized to day 10 (left axis) and CYP3A4 activity (measured as testosterone-6β-hydroxylation) and protein expression (right axis) are normalized to one million hepatocytes. Protein expressions were determined by mass spectrometry–determined abundance of peptide EVTNFLR relative to stable isotope (13C615N) labeled peptide used as an internal standard calibrator. Data shown are for one hepatocyte donor (3121A).
- Fig. 2.
P450 degradation of CYP3A4 as measured by SIL-containing peptide depletion for four donors with protein-specific peptides: (A) SLLSPTFTSGK, (B) EVTNFLR, (C) VEINGMFIPK, and (D) YWTEPEK. Labeled peptide abundances are expressed as percentages of the total measured peptide, n = 3 per time point with error bars representing S.D. Days are marked from the start of the HepatoPac cultures with SIL amino acids supplemented for days 0–10. Each line represents the linear regression for an individual donor from which the slope was used to calculate the protein t1/2.
Tables
- TABLE 1
Degradation rates (kdeg) and half-lives (t1/2) determined using HepatoPac-SILAC for CYP3A4
Data were calculated from four donors and are reported as mean and S.D. The average kdeg and t1/2 values determined for the three nonarginine-containing CYP3A4 peptides were 0.023 ± 0.002 h−1 and 29.7 ± 2.3 hours, respectively. The kdeg and t1/2 values for the arginine-containing peptide (EVTNFLR) were 0.033 ± 0.005 h−1 and 21.0 ± 2.6 hours, respectively. Recent kdeg and t1/2 value determinations for CYP3A4 using HepatoPac by mRNA following siRNA or interleukin 6 inhibition were 0.0240 h−1 and 28.9 hours, respectively (Ramsden et al., 2015); and by mRNA following induction they were 0.0261 h−1 and 26 hours, and 49 hours by activity following rifampicin induction (Dixit et al., 2016).
P450 Peptidea kdeg t1/2 h−1 h CYP3A4 SLLSPTFTSGK 0.023 ± 0.002 29.6 ± 1.9 CYP3A4 DVEINGMFIPK 0.023 ± 0.001 29.6 ± 1.0 CYP3A4 YWTEPEK 0.023 ± 0.001 29.8 ± 0.8 ↵a The SRM parameters of the specific peptides used for LC-MS/MS protein measurements are provided in Supplemental Table 2.
Data Supplement
Files in this Data Supplement:
- Supplemental Data -
Supplemental Table 1 - Donor information for hepatocytes used for HepatoPac cultures
Supplemental Table 2 - Summary of tryptic peptides and their selected reaction-monitoring parameters used for LC-MS/MS protein measurements
Supplemental Table 3 - CYP3A4 stability data (mRNA, protein expression, and enzymatic activity) for four HepatoPac donors for Days 10-13 of SILAC culture
Supplemental Table 4 - Calculated t1/2 (h) and kdeg (h-1) values from individual donors for CYP3
Supplemental Figure 1 - Representative SRM chromatograms showing CYP3A4 peptide, SLLSPTFTSGK, for one HepatoPac donor over Days 10-13 with SILAC
- Supplemental Data -
In this issue
Utility of SILAC in HepatoPac to Determine CYP3A4 kdeg
