In this project, we will develop novel quantitative high-throughput (HT) metabolic profiling methods and workflows that will facilitate the study and understanding of the dynamics of some of the building blocks of life (namely; metabolites, enzymes and cells), their interactions, and how their functioning is modulated by their environment. These novel HT workflows can be applied for a range of fields in the life sciences such as the characterization of enzyme activity, cell-based screening, drug research and future health care. HT analytical screening can accelerate and revolutionize research because (i) the availability of many quantitative time-resolved data points will lead to faster mechanistic understanding resulting in breakthroughs for development of new enzymes and cell systems, (ii) HT screening of cellular assays allows to obtain insights on cellular phenotypes, drug metabolism and transport, on/off target effects of compounds, etc. at a large scale and (iii) HT screening of organ-on-chip models or organoids provides insight in the dynamics and modulation of cellular molecular phenotypes due to exposure of drugs or stressors. Faster analytical data acquisition and analysis will allow faster novel design of experiments for the design-make-test-apply-learn cycle obtaining faster insights for product development . Automation of such an integrated workflow is essential to meet the HT requirements. The focus in this project is on HT sample generation and preparation, quantitative time-resolved HT metabolic profiling using mass spectrometry (MS), and proper data processing, handling and dynamic data analysis. Such sample- preparation workflows are often application-dependent and contain different steps, that should therefore be preferably modular to be integrated into the HT workflow. In recent years, Leiden University has been developing several innovative sample-preparation technologies that are in principle suited for a HT metabolic profiling workflow. These methods will be further optimized, extended, new interfaces made to couple them to each other, and integrated into a fully automated and miniaturized sample preparation workflow. Sample matrix will be removed, metabolites enriched in fractions, and then directly transferred to HT high resolution MS screening. Different (gentle) ionization options for MS will be compared and optimized to minimize degradation of labile compounds during ionization (so called in-source fragmentation). If samples are very complex, or different isomers have to be separated, fast separation in the liquid phase will be included, or ion-mobility separation and/or MS/MS analysis in the gas phase. Envisioned analysis times including sample preparation are sec’s for simpler samples and up to min’s for complex samples. The workflows developed in this project will run at the Leiden University and will be transferred (proof-of-concept) to the industrial partners within the consortium (DSM and AstraZeneca) via secondments and training sessions, enabling to speed up development of new or improved products of the different industrial partners. Interscience/SampleQ is interested to commercialize the automated sample-preparation workflow, Sciex is interested to develop and market HT MS metabolic profiling. Our research is expected to have significant utilization impact in a wide variety of application fields other than the ones specifically addressed in this proposal: the pharmaceutical HT (cell-based) metabolite workflows, represented by AstraZeneca, and the time-resolved HT metabolite workflow for extracellular and intracellular microbial enzymes, represented by DSM. Spin-off products of the HT MS metabolite workflow developed here are expected to be of interest for clinical and epidemiological studies, clinical disease assays, and metabolic health monitoring of the larger population