Absolute quantification of human uridine-diphosphate glucuronosyl transferase (UGT) enzyme isoforms 1A1 and 1A6 by tandem LC-MS.
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.
Division of Molecular Pharmaceutics, School of Pharmacy, University of North Carolina at Chapel Hill, NC 27599, USA.
John K
JK
Fallon
David E
DE
Harbourt
Saber H
SH
Maleki
Fay K
FK
Kessler
Joseph K
JK
Ritter
Philip C
PC
Smith
D000818:Animals; D015153:Blotting, Western; D002247:Carbon Isotopes; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D014453:Glucuronosyltransferase / Q000032:analysis*; D006801:Humans; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D009587:Nitrogen Isotopes; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000099148:UGT1A1 Enzyme
D002247:Carbon Isotopes; D014453:Glucuronosyltransferase; D009587:Nitrogen Isotopes; D000099148:UGT1A1 Enzyme; C110655:UDP-glucuronosyltransferase, UGT1A6