Absolute Quantification of Human Uridine-Diphosphate Glucuronosyl Transferase (UGT) Enzyme Isoforms 1A1 and 1A6 By Tandem LC-MS

Abstract

UGT enzymes catalyze the formation of glucuronic acid conjugates. Specifically selected representative stable isotope (C(13), N(15)) labeled peptide internal standards of each enzyme were employed to quantify UGTs 1A1 and 1A6 by LC-MS/MS using isotope dilution techniques. Inter day variability (n=5) for human liver microsomes was <or= 8.0 % for UGT1A1 and <or= 19 % for UGT1A6. Comparison within a human liver microsomal library showed a strong correlation with Western blot for UGT1A1 concentrations (r=0.988). The data presented indicates that an accurate and reproducible method for UGT absolute quantification can be established using LC-MS/MS analysis of characteristic peptides within the protein.

Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity

“…In-solution sample preparation was performed as reported previously (Fallon et al, 2008(Fallon et al, , 2013a. Briefly, sample protein content was assessed using bicinchoninic acid assay and samples (n = 60) were diluted 1:20 in 50 mM ammonium bicarbonate.…”

“…Selection of proteotypic peptides was carried out as previously reported (Fallon et al, 2008(Fallon et al, , 2013aKamiie et al, 2008). The peptides were tryptic, as well as proteotypic, and contained no linkers or added moieties, such that they could be used directly as standards for quantification.…”

“…Several studies published recently reported different proteomic methodologies driven by advances in LC-MS technology (Fallon et al, 2008;Ohtsuki et al, 2012;Achour et al, 2014a;Vildhede et al, 2014;Harwood et al, 2015;Fallon et al, 2016). These methodologies focused on obtaining expression values from a range of mammalian tissues/organs (e.g., liver, intestine, kidneys) and in vitro systems (e.g., hepatocytes, Caco-2 cell lines), with a view to providing systems data for in vitro-in vivo extrapolation (IVIVE) of pharmacokinetic profiles using computerized physiologically based pharmacokinetic (PBPK) models (Rostami-Hodjegan, 2012;Bosgra et al, 2014).…”

“…Individual enzyme mixtures also contained final substrate concentrations of 800 mM b-estradiol Details of theoretical selection criteria can be found in Fallon et al (2008), Kamiie et al (2008), and Pratt et al (2006).…”

Targeted Precise Quantification of 12 Human Recombinant Uridine-Diphosphate Glucuronosyl Transferase 1A and 2B Isoforms Using Nano-Ultra-High-Performance Liquid Chromatography/Tandem Mass Spectrometry with Selected Reaction Monitoring

“…Targeted isotope dilution techniques with tandem mass spectrometry have recently been used to quantify a wide range of bioactive proteins including UGTs, cytochrome P450s, and transporters (Li et al, 2009;Harbourt et al, 2012;Ohtsuki et al, 2012;Picotti et al, 2013). The specificity and broad dynamic range of the methods are advantageous when compared with often semiquantitative, nonspecific, and expensive traditional immunometric methods (Seppen et al, 1994;Ritter et al, 1999;Paine and Fisher, 2000;Fallon et al, 2008). In this study we present application of a previously described capillary liquid chromatography-tandem mass spectrometry isotope dilution method (Fallon et al, 2013) for the targeted quantification of up to 14 UGT isoforms to a series of commercially produced recUGT samples (BD Supersomes [baculovirus-infected insect-cell microsomes]) (12 isoforms; n = 49 samples).…”