Single-Cell Mass Spectrometry of Metabolites Extracted from Live Cells by Fluidic Force Microscopy

Abstract

Single-cell metabolite analysis provides valuable information on cellular function and response to external stimuli. While recent advances in mass spectrometry reached the sensitivity required to investigate metabolites in single cells, current methods commonly isolate and sacrifice cells, inflicting a perturbed state and preventing complementary analyses. Here, we propose a two-step approach that combines nondestructive and quantitative withdrawal of intracellular fluid with subpicoliter resolution using fluidic force microscopy, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The developed method enabled the detection and identification of 20 metabolites recovered from the cytoplasm of individual HeLa cells. The approach was further validated in C-glucose feeding experiments, which showed incorporation of labeled carbon atoms into different metabolites. Metabolite sampling, followed by mass spectrometry measurements, enabled the preservation of the physiological context and the viability of the analyzed cell, providing opportunities for complementary analyses of the cell before, during, and after metabolite analysis.

Spatial single-cell profiling of intracellular metabolomesin situ

“…The discovered critical roles of metabolism and the growing awareness of a hidden world beneath population averages created an urgent need to investigate intracellular metabolism at the single-cell level (Rubakhin et al, 2013; Zenobi, 2013). In the recent years, the sensitivity of mass spectrometry-based metabolite detection has improved substantially opening novel avenues to metabolomics of either single cells or small groups of cells (Do et al, 2017; Guillaume-Gentil et al, 2017; Ibáñez et al, 2013; Merrill et al, 2017) and even at a subcellular level (Passarelli et al, 2017). However, despite these methods successfully demonstrated detection of metabolites in individual cells, analytical and computational challenges precluded studies of spatio-molecular organization and cellular heterogeneity, and prevented discovering the links between intracellular metabolomes, cellular phenotypes and spatial organisation of cells.…”

Single‐Cell Mass Spectrometry Approaches to Explore Cellular Heterogeneity

“…[73] As uspension of S. cerevisiae cells was deposited on the platform, and single or af ew cells were trapped in each micro-well for MALDI-MS analysis.Metabolic variations between individual cells due to drug treatment or genetic modifications were explored, and phenotypic changes in metabolite levels were revealed. [80] Ac antilever probe,d riven by an atomic force microscope,w as used to gently extract 1t o3 pL of the cytoplasm from HeLa cells and dispense it onto the MAMS substrate for MALDI-MS analysis.A nother cell-trapping method, based on am icro-well patterned microfluidic chip, was introduced for the high-throughput analysis of phospholipids in single A549 cells by MALDI-MS imaging. [74,79] More recently,t his technique was combined with fluidic force microscopy (FluidFM) for the nondestructive and quantitative sampling of cell contents followed by MALDI-MS analysis (Figure 1).…”

“…[74,79] More recently,t his technique was combined with fluidic force microscopy (FluidFM) for the nondestructive and quantitative sampling of cell contents followed by MALDI-MS analysis (Figure 1). [80] Ac antilever probe,d riven by an atomic force microscope,w as used to gently extract 1t o3 pL of the cytoplasm from HeLa cells and dispense it onto the MAMS substrate for MALDI-MS analysis.A nother cell-trapping method, based on am icro-well patterned microfluidic chip, was introduced for the high-throughput analysis of phospholipids in single A549 cells by MALDI-MS imaging. [81] Another high-throughput MALDI-MS approach utilized microscope imaging to identify the coordinates of individual cells dispersed on amicroscope slide,and was used to analyze the peptide and protein species in targeted cells.…”

Intracellular Electrochemical Sensing

“…However, most microfluidic approaches do not provide the spatiotemporal information on the intracellular contents because the collection methods are based on complete cell lysis. To study the dynamics of intracellular transportation or localization of cytoplasmic content , techniques for extracting subcellular cytoplasm are needed. In this section, we focused on recent electrical techniques of collecting subcellular cytoplasm (Figure ).…”