Thurs Sept 21, 2023 – MSDG Vendor Show & Symposium

Knowledge of all the types of molecules that are produced in cells is key to future discoveries. For example, understanding cell differentiation during early patterning of the chordate embryo and its brain regions can provide insights into the treatment of various developmental diseases. How functionally important molecules, such as proteins and metabolites, contribute to these cell processes is largely unknown. The limitation has been a lack of sufficiently sensitive mass spectrometry technologies that can measure these biomolecules with compatibility for live development, a prerequisite for functional biology. In this presentation, we will discuss the development of in situ/vivo approaches by cellular and subcellular mass spectrometry that enabled our lab to determine the proteomic and metabolomic profile of identified cells in live Xenopus laevis frog embryos developing to tadpoles and neurons in mouse brain tissues. Molecular measurements using capillary electrophoresis time-of-flight or orbitrap mass spectrometer platforms revealed proteomic and metabolomic differences between the X. laevis cells that correlated with phenotype. Follow-up functional experiments led to discoveries of molecules capable of altering normal cell fate decisions in the chordate embryo. The technology was extended to smaller cells, neurons in the mouse brain. Quantification of ~250–800 different proteins among three different types of neurons revealed reproducible proteomic differences between the neuron types. Capillary electrophoresis mass spectrometry expands the molecular toolbox of cell biology and neuroscience.

Drug metabolizing enzymes and transporters are critical determinants of drug absorption, metabolism, distribution, and elimination (ADME) and influence drug-drug interactions (DDIs) and response. While significant progress has been made to utilize in vitro models to predict drug ADME using physiologically-based pharmacokinetic (PBPK) models, these models require comprehensive physiological data on inter-individual variability. In particular, PBPK models require quantitative information on the levels and activity of individual pathways involved in drug disposition across different tissues and populations (healthy vs. diseased or children vs. adults). A significant lack of quantitative knowledge regarding non-Cytochrome P450 (non-CYP) enzymes and transporters in human is the major limitation towards building PBPK models in the drug development which often results inaccurate in vitro to in vivo extrapolation (IVIVE) and poor prediction of interindividual variability of drug metabolism. Although non-CYP enzymes are expressed in multiple human tissues, differential tissue expression and interindividual variability in the expression of these enzymes are not well studied. Uncharacterized sub-cellular localization of some non-CYP enzymes is another knowledge gap with respect to the development of quantitatively viable in vitro and in silico models. Similarly, animal to human scaling of non-CYP metabolism is not accurate because of the unknown inter-species differences. To address these issues, we utilize state-of-the-art quantitative proteomics in conjunction with metabolomics and genomics approaches to characterize abundances and activity of drug metabolizing enzymes and transporters in human tissues and biofluids. These data are then integrated into PBPK models to predict variability in drug clearance and DDIs, particularly in underrepresented populations such as children in whom clinical studies are not routinely performed.