Targeted Quantitative Proteomics for the Analysis of 14 UGT1As and -2Bs in Human Liver Using NanoUPLC–MS/MS with Selected Reaction Monitoring

Abstract

Targeted quantitative proteomics using heavy isotope dilution techniques is increasingly being utilized to quantify proteins, including UGT enzymes, in biological matrices. Here we present a multiplexed method using nanoLC-MS/MS and multiple reaction monitoring (MRM) to quantify 14 UGT1As and UGT2Bs in liver matrices. Where feasible, we employ two or more proteotypic peptides per protein, with only four proteins quantified with only one proteotypic peptide. We apply the method to analysis of a library of 60 human liver microsome (HLM) and matching S9 samples. Ten of the UGT isoforms could be detected in liver, and the expression of each was consistent with mRNA expression reported in the literature. UGT2B17 was unusual in that ∼30% of liver microsomes had no or little (<0.5 pmol/mg protein) content, consistent with a known common polymorphism. Liver S9 UGT concentrations were approximately 10-15% those of microsomes. The method was robust, precise, and reproducible and provides novel UGT expression data in human liver that will benefit rational approaches to evaluate metabolism in drug development.

Quantitative Targeted Proteomics for Membrane Transporter Proteins: Method and Application

“…Followed by the selection of quantitative targeted peptides, the optimization of SRM, the selection of internal standards, and sample digestions, sample analysis can be conducted on a triple quadrupole mass spectrometer such as API 4000 or 6500 coupled to an LC, ultra performance LC (UPLC), or nano-UPLC system (22,23,37). The LC separation can be achieved in a reverse phase column (e.g., 100× 3.0 mm particle size 2.6 μm Kinetex® C18 column) with a gradient elution of water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B) as mobile phases.…”

“…To monitor multiple proteins in a single run, scheduled MRM acquisition method could be constructed using manually optimized declustering potentials, collision energies, as well as collision cell entrance and exit potentials. Three MRMs can be monitored simultaneously for each peptide (22,23). The quality control (QC) should also be conducted for the parameters of bioanalytical method validation guidelines suggested by regulatory agency for peptide stability, selectivity, linearity, reproducibility, and intra-/ interday variability (55).…”

“…The limitations of this approach include the need for high end instruments and potential loss of protein mapping coverage due to low abundance of membrane protein expression. As mentioned above, upfront sample enrichment and use of nano-flow LC instead of regular LC could improve protein mapping coverage as well as peptide detection sensitivity (18,23).…”

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

“…All other reagents and solvents used were from standard suppliers and were of reagent or HPLC grade with all purities as defined by the manufacturer. Materials and chemicals used for the quantification of UGT abundances were as described previously (Fallon et al, 2013b;Achour et al, 2014a).…”

Applications of targeted proteomics in systems biology and translational medicine

“…Shi and coworkers applied the PRISM targeted proteomics to quantitate AGR2 in human urine at serum at concentrations of approximately 130 pg/ml and 10 pg per 100 ug of total protein mass in urine, respectively, and found in a proof‐of‐concept study of 37 urine samples that AGR2/PSA concentration ratios can distinguish noncancer and cancer 85. Fallon and coworkers developed assays for 14 UGT1As and UGT2Bs across 60 human liver microsomes and matching S9 samples to evaluate metabolism in drug development 86.…”