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.