Abstract
Quantification methods employing stable isotope-labeled peptide standards and liquid chromatography–tandem mass spectrometry are increasingly being used to measure enzyme amounts in biologic samples. Isoform concentrations, combined with catalytic information, can be used in absorption, distribution, metabolism, and excretion studies to improve accuracy of in vitro/in vivo predictions. We quantified isoforms of uridine-diphosphate glucuronosyltransferase (UGT) 1A and 2B in 12 commercially available recombinant UGTs (recUGTs) (n = 49 samples) using nano-ultra-high-performance liquid chromatography–tandem mass spectrometry with selected reaction monitoring). Samples were trypsin-digested and analyzed using our previously published method. Two MRMs were collected per peptide and averaged. Where available, at least two peptides were measured per UGT isoform. The assay could detect UGTs in all recombinant preparations: recUGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17, with limit of detection below 1.0 pmol/mg protein for all isoforms. The assay had excellent linearity in the range observed (2–15.5 pmol/mg, after dilution). Examples of concentrations determined were 1465, 537, 538, 944, 865, 698, 604, 791, 382, 1149, 307, and 740 pmol/mg protein for the respective isoforms. There was a 6.9-fold difference between the maximum and minimum recUGT concentrations. The range of concentrations determined indicates that catalytic rates per mg total protein in vitro will not accurately reflect isoform inherent specific activity for a particular drug candidate. This is the first report of a targeted precise quantification of commercially available recUGTs. The assay has potential for use in comparing UGT amounts with catalytic activity determined using probe substrates, thus allowing representation of catalysis as per pmol of UGT isoform.
Footnotes
- Received July 23, 2013.
- Accepted September 12, 2013.
The project was supported in part by Pfizer, Inc., and an instrumentation grant from the National Institutes of Health [Grant S10, RR024595].
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics
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