Project Summary Fundamental understanding of the development of cellular metabolic heterogeneity and its impact on cell response to stimuli has ramifications on nearly every aspect of biomedical research including disease diagnosis, treatment and prevention. Cellular metabolism is naturally heterogeneous and prediction of cellular response to stimuli will depend on the distribution of intracellular metabolite concentrations across a cell population. However, for small molecules and therapeutics direct empirical measure of intracellular concentrations is rare, though many drugs target inside the cell and require reaching their site of action at a specific concentration to be effective. Their quantitative distribution in a cell population is a key, currently unmeasurable, variable to understanding many drug’s pharmacological and toxicological effects. The best way to deconvolute heterogeneous cell response is to individually profile each cell in the population. Unfortunately, current single cell analysis approaches for measuring small molecules lack a number of critical features needed to make analysis routine. An ideal single cell characterization method must be quantitative, fast, robust, sensitive, applicable to a range of cell types and be able to measure a wide range of molecules. The lack of such a system restricts detailed investigations into the fundamental mechanisms underpinning heterogeneous cell response; foundational information needed for advancing disease diagnosis, treatment and prevention as part of the mission of National Institute of General Medical Sciences (NIGMS). The goal of this research is to evaluate a high-risk/high-reward approach, pulsed single cell mass spectrometry (p-SC-MS), a novel concept of capturing, lysing and analyzing single cells with significantly improved sensitivity and throughput over existing single cell analysis methodologies for small molecules. Our approach requires overcoming high-risk challenges in droplet capture and cell lysis at small scale and under a high electric field environment. This risk is offset by a correspondingly high reward: massively increased sensitivity (>50,000X), single cell analysis throughput (>6X) and cell media tolerance over the current state-of- the-art. The platform is easy to use and will be shown to be applicable for many cell types through three specific aims tackling the feasibility of the concept. Specific Aim #1: Demonstrate single cell droplet-on-demand capture and metabolite extraction using a custom pulsed-nanoelectrospray ionization source. Specific Aim #2: Validate the quantitative accuracy of p-SC-MS through measure of amiodarone (AMIO) and N-desethylamiodarone (NDEA) in single HepG2 cells. Specific Aim #3: Demonstrate measure and comparison of metabolites and lipids in HeLa, HepG2, MD-AMB, BT-474 and OK-F6 cell lines in complex media. The end product of this research will be a novel, high-impact technology that enables chemical characterization of small molecules from single cells with a massive >50,000X improvement in sensitivity and 6X improvement in speed over current art, enabling resolution of intracellular molecular distributions.

Project Narrative The mechanisms underpinning heterogeneous metabolic response between individual cells remain poorly understood and current single cell analysis approaches for measuring metabolites in individual cells have several technical limitations that impede basic research in this area. We are proposing a high-risk/high-reward approach, pulsed single cell mass spectrometry (p-SC-MS), a novel concept of capturing, lysing and analyzing single cells at small scale with a massively improved sensitivity and throughput over existing single cell analysis methodologies for small molecules. The goal of this proposal is to rigorously evaluate p-SC-MS so that it can become an effective research tool in the biomedical community and provide insights into the fundamental mechanisms underpinning heterogeneous cell response; foundational information needed for advancing disease diagnosis, treatment and prevention.