Description of an LC-MS Technique for Studying Substrate, Metabolite And Enzyme Concentrations From the Same Samples

Hypothesis: A combination of targeted proteomics tools and workflows and traditional small mol-ecule LC-MS methods can be used to evaluate the concentrations of enzymes, substrates, and metabolites from the same sample.
Methods: Substrate and metabolite concentrations are determined in a traditional small molecule LC-MS/MS bioanlaytical workflow. We are using a Qtrap 5500 from AB Sciex coupled to an Eksi-gent microflow LC running 1.0 mm ID columns at 50 ul/min. The column is an Eksigent 3C18-CL-300, 3 um, 300A, 1mm x 50mm. Gradients are somewhat dependent on the system under study, but all are acetonitrile and water gradients running between 5 minutes and 10 minutes. Sample preparation is simple protein precipitation using acidified acetonitrile and centrifugation.
Enzyme concentrations are determined by using a surrogate peptide approach where tryptic pep-tides are used as markers of the entire protein1. The tryptic peptides are quantified using LC-MS/MS or MRM3 on the Qtrap5500. Calibration standards are generated from either purified protein or synthetic stable isotope labeled tryptic peptides of known concentration and purity.
Preliminary Data: We have investigated the feasibility of the technique using UGT enzymes in microsomes from recombinant over-expressing cell systems (BD Biosciences) and liver (BD Bio-sciences). The data collected to date is from UGT 2B7 using AZT as a substrate for conjugation. 50 individual human liver microsome samples were evaluated for activity and protein concentra-tion in separate events. A small subset of 10 individuals will be evaluated in the combined work-flow
Discussion: The work presented here is aimed at developing LC-MS/MS methods for the quanti-tative determination of small molecules and proteins in individual samples. We have adapted the targeted proteomics tools for mass spectrometry and combined them with the widely used LC-MS approaches for quantification of drugs and metabolites to generate methods capable of measur-ing the concentrations of enzyme, substrate and metabolite in the same sample over relatively large numbers of samples. The presentation will cover the procedures followed, the inherent limi-tations of the technique that may affect interpretation of the results relative to the enzyme kinetics, and a simple example using recombinant cell expression systems and liver microsomes to demonstrate the feasibility of the approach.


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