PROJECT SUMMARY Investigations of Proteome Turnover Kinetics Under Cellular Differentiation: Proteins are in a constant flux of continuous synthesis and degradation, both of which contribute to regulate protein levels in the cell. Although this fact has been known for 80 years, currently most biological inquiries are limited to static snapshots of overall transcript and protein levels, whereas knowledge into the dynamic changes of protein turnover remains severely lagging. This ESI MIRA proposal seeks to advance the current understanding of how cellular proteomes remodel during cell state transitions, by incorporating protein turnover kinetics information at key stages of human induced pluripotent stem cell (iPSC) differentiation into distinct cellular lineages. Working with collaborators, we previously developed deuterium stable isotope labeling, mass spectrometry, and kinetic modeling methods to quantitate protein turnover in animal models and in human. In doing so, we found many novel cell states and disease markers may be discovered from integrating orthogonal protein abundance and kinetics information. In the next five years, we propose to: (1) apply these methods to acquire a high temporal density map of human iPSC trilineage differentiation into cardiomyocyte, hepatocyte, and neuroprogenitor cells; (2) interrogate the regulatory principles that govern turnover flux across different differentiation stages; and (3) assess the functional consequences of protein degradation on the success and cell maturity of terminal cell production. Finally, a limitation of current techniques is that the kinetic models used in protein turnover studies largely assume a non-changing protein pool size at steady state, which does not apply to differentiating cells or progressing diseases. We propose to expand current models of analyzing heavy water stable isotope label experiments toward dynamical systems with variable protein pool sizes. If successful, the proposed research would greatly expand the current knowledge of molecular events that take place during human iPSC differentiation, as well as generate publicly available data sets and software tools to advance protein turnover studies in diverse areas.

PROJECT NARRATIVE The composition and concentration of proteins in a cell are tightly regulated to maintain cell identity and ensure physiological functions. We propose to apply stable isotope labeling, mass spectrometry, and kinetic modeling methods to understand how the processes of protein synthesis and degradation are orchestrated during cell state transitions in human induced pluripotent stem cell differentiation into various cell types. The resulting knowledge, data, and software tools have the potential to advance current understanding of cellular differentiation and open new avenues for disease modeling and tissue engineering applications.