Absolute quantification of UGT1A1 in various tissues and cell lines using isotope label-free UPLC-MS/MS method determines its turnover number and correlates with its glucuronidation activities

Abstract

Uridine 5'-diphosphate-glucuronosyltransferase (UGT)1A1 is a major phase II metabolism enzyme responsible for glucuronidation of drugs and endogenous compounds. The purpose of this study was to determine the expression level of UGT1A1 in human liver microsomes and human cell lines by using an isotope label-free LC-MS/MS method. A Waters Ultra performance liquid chromatography (UPLC) system coupled with an API 5500Qtrap mass spectrometer was used for the analysis. Two signature peptides (Pep-1, and Pep-2) were employed to quantify UGT1A1 by multiple reaction monitoring (MRM) approach. Standard addition method was used to validate the assay to account for the matrix effect. 17β-Estradiol was used as the marker substrate to determine UGT1A1 activities. The validated method has a linear range of 200-0.0195nM for both signature peptides. The precision, accuracy, and matrix effect were in acceptable ranges. UGT1A1 expression levels were then determined using 8 individual human liver microsomes, a pooled human liver microsomes, three UGT1A1 genotyped human liver microsomes, and four cell lines (Caco-2, MCF-7, Hela, and HepG2). The correlations study showed that the UGT1A1 protein levels were strongly correlated with its glucuronidation activities in human liver microsomes (R(2)=0.85) and in microsomes prepared from cell lines (R(2)=0.95). Isotope-labeled peptides were not necessary for LC-MS/MS quantitation of proteins. The isotope label-free absolute quantification method used here had good accuracy, sensitivity, linear range, and reproducibility, and were used successfully for the accurate determination of UGT1A1 from tissues and cell lines.

Keywords: Absolute quantification; CE; CXP; CYP; DP; Glucuronidation; HLM; LC–MS/MS; LLOQ; MRM; Microsomes; PTM; RLM; UGT; UGT1A1; UPLC; collision cell exit potential; collision energy; cytochrome P450; declustering potential; human liver microsome; lower limit of quantification; multiple reaction monitoring; post translational modification; rat liver microsome; ultra performance liquid chromatography; uridine 5′-diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase).

Fig. 1

(A) MS/MS spectra of m/z 457.9, +2-charged DGAFYTLK and m/z 504.4, +2-charged TYPVPFQR obtained from ESI-MS in positive ionization mode. Arrows indicate optimum product ions for use in MRM transitions. (B) The table shows the MRM transitions (Q1/Q3 settings) selected for the tryptic peptides.

Fig. 2

MRM chromatograms of tryptic peptides derived from human UGT1A1 supersome™ showing chromatograms of two tryptic peptides were detected by four MRM transitions, eluting at 2.5 min (A, for microsome sample) and 4.9 min (B, for cell samples).

Fig. 3

Optimization of sample preparation. (A) Time course study of trypsin digestion: the peak area for the peptides with m/z 457.9 and 504.4 are plotted against the reaction time. (B) Matrix effect was investigated by comparing the peak area and slope of calibration curves between two different sets of calibration curves. The absence or presence of matrix effect on the quantification was assessed. Dots denote four continuous diluted calibrants in 0.1% formic acid; Circles denote four continuous diluted calibrants digested with 200 μg rat liver microsomes (RLM).

Fig. 4

Validation for LC-MS/MS quantification of UGT1A1 protein using standard addition method. (A) Shown were overlaps of three MRM chromatograms of a (digested) individual HLM, standard calibrant (12.5 nM), and the digested HLM spiked with the calibrant (12.5nM). The accuracy and coefficient of variation (CV%) of pHLM, HLM 442, HLM 743, and HLM751 were analyzed and shown in table. (B) The standard addition method was also performed for validation of UGT1A1 quantification in cell microsomal fraction. Three overlapped MRM chromatograms are from digested Caco-2 cell microsomes, standard calibrant (0.78 nM), and digested Caco-2 cell microsomes spiked with calibrant (0.78 nM). The table shows value of accuracy and CV for Caco-2, Hela, MCF-7, and HepG2 cells. The data represents the mean± SD (n=3). (C) Plots of standard addition curve for digested cell microsomal protein derived from Caco-2, HeLa, MCF-7 and HepG2 cell. The standard addition experiment was conducted by spiking digested 50 μg cell microsomal protein with a dilution series of calibrants 0.78 nM, 1.56 nM, 3.125 nM and 6.25 nM (Curve for Hela and MCF-7). The regression equantions and R² values were presented in the inset.

Fig. 4

Validation for LC-MS/MS quantification of UGT1A1 protein using standard addition method. (A) Shown were overlaps of three MRM chromatograms of a (digested) individual HLM, standard calibrant (12.5 nM), and the digested HLM spiked with the calibrant (12.5nM). The accuracy and coefficient of variation (CV%) of pHLM, HLM 442, HLM 743, and HLM751 were analyzed and shown in table. (B) The standard addition method was also performed for validation of UGT1A1 quantification in cell microsomal fraction. Three overlapped MRM chromatograms are from digested Caco-2 cell microsomes, standard calibrant (0.78 nM), and digested Caco-2 cell microsomes spiked with calibrant (0.78 nM). The table shows value of accuracy and CV for Caco-2, Hela, MCF-7, and HepG2 cells. The data represents the mean± SD (n=3). (C) Plots of standard addition curve for digested cell microsomal protein derived from Caco-2, HeLa, MCF-7 and HepG2 cell. The standard addition experiment was conducted by spiking digested 50 μg cell microsomal protein with a dilution series of calibrants 0.78 nM, 1.56 nM, 3.125 nM and 6.25 nM (Curve for Hela and MCF-7). The regression equantions and R² values were presented in the inset.

Fig. 4

Validation for LC-MS/MS quantification of UGT1A1 protein using standard addition method. (A) Shown were overlaps of three MRM chromatograms of a (digested) individual HLM, standard calibrant (12.5 nM), and the digested HLM spiked with the calibrant (12.5nM). The accuracy and coefficient of variation (CV%) of pHLM, HLM 442, HLM 743, and HLM751 were analyzed and shown in table. (B) The standard addition method was also performed for validation of UGT1A1 quantification in cell microsomal fraction. Three overlapped MRM chromatograms are from digested Caco-2 cell microsomes, standard calibrant (0.78 nM), and digested Caco-2 cell microsomes spiked with calibrant (0.78 nM). The table shows value of accuracy and CV for Caco-2, Hela, MCF-7, and HepG2 cells. The data represents the mean± SD (n=3). (C) Plots of standard addition curve for digested cell microsomal protein derived from Caco-2, HeLa, MCF-7 and HepG2 cell. The standard addition experiment was conducted by spiking digested 50 μg cell microsomal protein with a dilution series of calibrants 0.78 nM, 1.56 nM, 3.125 nM and 6.25 nM (Curve for Hela and MCF-7). The regression equantions and R² values were presented in the inset.

Fig. 5

(A) Comparison of human UGT1A1 protein level measured by LC-MS/MS in digested different human tissue micrsomal samples. Peptide concentrations were converted to pmol UGT1A1 protein /mg microsomal protein. The Blank bars and solid bars denote the protein level of UGT1A1 obtained in the present study were determined using two MRM transitions respectively. Data are presented as mean± SD (n=3). (B) Correlation between the protein levels of UGT1A1 and 17β-estradiol glucuronidation. The eight individual human liver microsomes (HLMs) were indicated in rhombus. Pooled liver microsomes (pHLM) and UGT1A1 genotyped HLMs were indicated in triangle and circles, respectively. R² values are Pearson’s correlation coefficient.

Fig. 6

(A) Absolute quantification of human UGT1A1 protein in human derived cell lines. The microsomal protein prepared from Caco-2, HeLa, MCF-7, and HepG2 cells were subjected to LC-MS/MS analysis for absolute quantification of UGT1A1 protein. The blank and solid bars indicate UGT1A1 protein level (pmol/mg protein) determined by two MRM transitions from peptide 1 and peptide 2 respectively. The data represents the mean± SD (n=3). The table below histogram gives the corresponding number. (B) Correlation between the protein levels of UGT1A1 and 17β-estradiol glucuronidation in human derived cell lines. Microsomal proteins isolated from Caco-2, HeLa, MCF-7, and HepG2 cells were used in the present study. R² values are Pearson’s correlation coefficient.