Metabolites In Single Cells Research Articles

Quantitative Single-Cell Mass Spectrometry Provides a Highly Resolved Analysis of Natural Product Biosynthesis Partitioning in Plants.

Plants produce an extraordinary array of natural products (specialized metabolites). Notably, these structurally complex molecules are not evenly distributed throughout plant tissues but are instead synthesized and stored in specific cell types. Elucidating both the biosynthesis and function of natural products would be greatly facilitated by tracking the location of these metabolites at the cell-level resolution. However, detection, identification, and quantification of metabolites in single cells, particularly from plants, have remained challenging. Here, we show that we can definitively identify and quantify the concentrations of 16 molecules from four classes of natural products in individual cells of leaf, root, and petal of the medicinal plant Catharanthus roseus using a plate-based single-cell mass spectrometry method. We show that identical natural products show substantially different patterns of cell-type localization in different tissues. Moreover, we show that natural products are often found in a wide range of concentrations across a population of cells, with some natural products at concentrations of over 100 mM per cell. This single-cell mass spectrometry method provides a highly resolved picture of plant natural product biosynthesis partitioning at a cell-specific resolution.

High-Throughput Metabolite Analysis of Unicellular Microalgae by Orthogonal Hybrid Ionization Label-Free Mass Cytometry.

Microalgae metabolite analysis is fundamental for the rational design of metabolic engineering strategies for the biosynthesis of high-value products. Mass spectrometry (MS) has been utilized for single-cell microalgae analysis. However, limitations in the detection throughput and polarities of detectable substances make it difficult to realize high-throughput screening of high-performance microalgae. Herein, a plasma-assisted label-free mass cytometry, named as PACyESI-MS, was proposed combining the advantages of orthogonal hybrid ionization and high-throughput MS analysis, which realized rapid metabolite detection of single microalgae. The cell detection throughput of PACyESI-MS was up to 52 cells/min. Dozens of the critical primary and secondary metabolites within single microalgae were detected simultaneously, including pigments, lipids, and energy metabolites. Furthermore, metabolite changes of Chlamydomonas reinhardtii and Haematococcus pluvialis under nitrogen deficiency stress were studied. Discrimination of Chlamydomonas under different nutrient conditions was realized using single-cell metabolite profiles obtained by PACyESI-MS. The relationships between the accumulation of bioactive astaxanthin and changes in functional primary metabolites of Haematococcus were investigated. It was demonstrated that PACyESI-MS can detect the flexible change of metabolites in single microalgae cells under different nutritional conditions and during the synthesis of high-value products, which is expected to become an important tool for the design of metabolic engineering-based high-performance microalgae factories.

Single-cell metabolite profiling enables information-rich classification of lymphocyte types and subtypes.

Lymphocytes play crucial roles in the human immune system; however, detailed metabolite characteristics need to be further investigated. Herein, we propose a lymphocyte classification method based on metabolite profiling at the single-cell level. The percentages of different lymphocyte types were calculated with a low margin of error, confirming that the metabolites could serve as a basis for lymphocyte classification. Furthermore, we analyzed the CD4/CD8 ratio in human peripheral blood to verify the feasibility of this method for the classification of lymphocyte subtypes. The proposed method is expected to be a potential tool for the clinical diagnosis of lymphocyte-related diseases.

Single-cell metabolic fingerprints discover a cluster of circulating tumor cells with distinct metastatic potential

Circulating tumor cells (CTCs) are recognized as direct seeds of metastasis. However, CTC count may not be the “best” indicator of metastatic risk because their heterogeneity is generally neglected. In this study, we develop a molecular typing system to predict colorectal cancer metastasis potential based on the metabolic fingerprints of single CTCs. After identification of the metabolites potentially related to metastasis using mass spectrometry-based untargeted metabolomics, setup of a home-built single-cell quantitative mass spectrometric platform for target metabolite analysis in individual CTCs and use of a machine learning method composed of non-negative matrix factorization and logistic regression, CTCs are divided into two subgroups, C1 and C2, based on a 4-metabolite fingerprint. Both in vitro and in vivo experiments demonstrate that CTC count in C2 subgroup is closely associated with metastasis incidence. This is an interesting report on the presence of a specific population of CTCs with distinct metastatic potential at the single-cell metabolite level.

Real-Time Assessment of Intracellular Metabolites in Single Cells through RNA-Based Sensors.

Quantification of the concentration of particular cellular metabolites reports on the actual utilization of metabolic pathways in physiological and pathological conditions. Metabolite concentration also constitutes the readout for screening cell factories in metabolic engineering. However, there are no direct approaches that allow for real-time assessment of the levels of intracellular metabolites in single cells. In recent years, the modular architecture of natural bacterial RNA riboswitches has inspired the design of genetically encoded synthetic RNA devices that convert the intracellular concentration of a metabolite into a quantitative fluorescent signal. These so-called RNA-based sensors are composed of a metabolite-binding RNA aptamer as the sensor domain, connected through an actuator segment to a signal-generating reporter domain. However, at present, the variety of available RNA-based sensors for intracellular metabolites is still very limited. Here, we go through natural mechanisms for metabolite sensing and regulation in cells across all kingdoms, focusing on those mediated by riboswitches. We review the design principles underlying currently developed RNA-based sensors and discuss the challenges that hindered the development of novel sensors and recent strategies to address them. We finish by introducing the current and potential applicability of synthetic RNA-based sensors for intracellular metabolites.

Dynamic fluctuations in a bacterial metabolic network

The operation of the central metabolism is typically assumed to be deterministic, but dynamics and high connectivity of the metabolic network make it potentially prone to generating fluctuations. However, time-resolved measurements of metabolite levels in individual cells that are required to characterize such fluctuations remained a challenge, particularly in small bacterial cells. Here we use single-cell metabolite measurements based on Förster resonance energy transfer, combined with computer simulations, to explore the real-time dynamics of the metabolic network of Escherichia coli. We observe that steplike exposure of starved E. coli to glycolytic carbon sources elicits large periodic fluctuations in the intracellular concentration of pyruvate in individual cells. These fluctuations are consistent with predicted oscillatory dynamics of E. coli metabolic network, and they are primarily controlled by biochemical reactions around the pyruvate node. Our results further indicate that fluctuations in glycolysis propagate to other cellular processes, possibly leading to temporal heterogeneity of cellular states within a population.

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Polarity-Specific Profiling of Metabolites in Single Cells by Probe Electrophoresis Mass Spectrometry.

Sensitive analysis of metabolites in a single cell is of fundamental significance for the better understanding of biological variability, differential susceptibility in disease therapy, and cell-to-cell heterogeneity as well. Herein, polarity-specific profiling of metabolites in a single cell was implemented by probe electrophoresis mass spectrometry (PEMS), which combined electrophoresis sampling of metabolites from a single cell and nanoelectrospray ionization-mass spectrometry (nanoESI-MS) analysis of the sampled metabolites. Enhanced extraction of either negatively or positively charged metabolites from a single cell was achieved by applying a DC voltage offset of +2.0 and -2.0 V to the probe, respectively. The experimental data demonstrated that PEMS features high throughput (≥200 peaks) and high sensitivity (≥10-times signal enhancement for [Choline + H]+, [Glutamine + H]+, [Arginine + H]+, etc.) in comparison with direct nanoESI-MS analysis. The biological effects of CdSe quantum dots (QDs) and γ-radiation on Allium cepa cells were investigated by PEMS, which revealed that CdSe QDs lead to the increase of intracellular amines while γ-radiation causes the decrease of intracellular acids. Therefore, this work provides an alternative platform to probe novel insights of cells by sensitive analysis of polarity-specific metabolites in a single cell.

Single-cell metabolite detection and genomics reveals uncultivated talented producer.

The production of bioactive metabolites is increasingly recognized as an important function of host-associated bacteria. An example is defensive symbiosis that might account for much of the chemical richness of marine invertebrates including sponges (Porifera), 1 of the oldest metazoans. However, most bacterial members of sponge microbiomes have not been cultivated or sequenced, and therefore, remain unrecognized. Unequivocally linking metabolic functions to a cellular source in sponge microbiomes is, therefore, a challenge. Here, we report an analysis pipeline of microfluidic encapsulation, Raman microscopy, and integrated digital genomics (MERMAID) for an efficient identification of uncultivated producers. We applied this method to the chemically rich bacteriosponge (sponge that hosts a rich bacterial community) Theonella swinhoei, previously shown to contain 'Entotheonella' symbionts that produce most of the bioactive substances isolated from the sponge. As an exception, the antifungal aurantosides had remained unassigned to a source. Raman-guided single-bacterial analysis and sequencing revealed a cryptic, distinct multiproducer, 'Candidatus Poriflexus aureus' from a new Chloroflexi lineage as the aurantoside producer. Its exceptionally large genome contains numerous biosynthetic loci and suggested an even higher chemical richness of this sponge than previously appreciated. This study highlights the importance of complementary technologies to uncover microbiome functions, reveals remarkable parallels between distantly related symbionts of the same host, and adds functional support for diverse chemically prolific lineages being present in microbial dark matter.

The Intriguing Landscape of Single-Cell Protein Analysis.

Profiling protein expression at single‐cell resolution is essential for fundamental biological research (such as cell differentiation and tumor microenvironmental examination) and clinical precision medicine where only a limited number of primary cells are permitted. With the recent advances in engineering, chemistry, and biology, single‐cell protein analysis methods are developed rapidly, which enable high‐throughput and multiplexed protein measurements in thousands of individual cells. In combination with single cell RNA sequencing and mass spectrometry, single‐cell multi‐omics analysis can simultaneously measure multiple modalities including mRNAs, proteins, and metabolites in single cells, and obtain a more comprehensive exploration of cellular signaling processes, such as DNA modifications, chromatin accessibility, protein abundance, and gene perturbation. Here, the recent progress and applications of single‐cell protein analysis technologies in the last decade are summarized. Current limitations, challenges, and possible future directions in this field are also discussed.

Probing the formation of anhydrovinblastine in Catharanthus roseus by single-cell mass spectrometry

Anhydrovinblastine, derived from the periwinkle plant Catharanthus roseus, is the direct precursor of vinblastine that is an important drug to treat a number of types of cancer. Insights into the formation mechanism of anhydrovinblastine at the cell level is beneficial for improving the industrial production of anticancer drugs. Herein, metal microneedle was modified for extracting single-cell metabolites of Catharanthus roseus, and then the microneedle was directly served as emitter of electrospray ionization for direct mass spectrometry analysis of single-cell metabolites. Sampling efficiency of microneedle with different surface modifications were compared. Different cells in stem, root, and leaf of Catharanthus roseus were also investigated. Our results showed that two key monomeric precursor molecules, vindoline and catharanthine, are from different cells in Catharanthus roseus and could mix to form anhydrovinblastine. This work shows a new method for probing the biosynthesis and formation of bioactive molecules in living systems.

Single Cell Mass Spectrometry With a Robotic Micromanipulation System for Cell Metabolite Analysis

The increasing demand for unraveling cellular heterogeneity has boosted single cell metabolomics studies. However, current analytical methods are usually labor-intensive and hampered by lack of accuracy and efficiency. we developed a first-ever automated single cell mass spectrometry system (named SCMS) that facilitated the metabolic profiling of single cells. In particular, extremely small droplets of sub nano-liter were generated to extract the single cells, and the underlying mechanism was verified theoretically and experimentally. This was crucial to minimize the dilution of the trace cellular contents and enhance the analytical sensitivity. Based on the precise 3D positioning of the pipette tip, we established a visual servoing robotic micromanipulation platform on which single cells were sequentially extracted, aspirated, and ionized, followed by the mass spectrometry analyses. With the SCMS system, inter-operator variability was eliminated and working efficiency was improved. The performance of the SCMS system was validated by the experiments on bladder cancer cells. MS and MS2 analyses of single cells enable us to identify several cellular metabolites and the underlying inter-cell heterogeneity. In contrast to traditional methods, the SCMS system functions without human intervention and realizes a robust single cell metabolic analysis. the SCMS system upgrades the way how single cell metabolites were analyzed, and has the potential to be a powerful tool for single cell metabolomics studies.

Nanothorn Filter-Facilitated Online Cell Lysis for Rapid and Deep Intracellular Profiling by Single-Cell Mass Spectrometry.

Mass spectrometry combined with flow cytometry is emerging for high-throughput single-cell metabolite analysis but still has problems with limited intracellular information coverage. Here, we show a simple and efficient all-in-one system integrating cell injection, cell extraction, online cell lysis, analyte ionization, and mass spectrometric detection for rapid single-HeLa-cell screening with in-depth profiling of cellular metabolites and drugs. Zinc oxide nanothorn-decorated filters with three bore sizes (5.22, 8.36, and 16.75 μm) were fabricated for efficient online lysis of the cell membrane (even nuclear membrane) to facilitate intracellular analyte release and demonstrated to have a size effect for potential subcellular discrimination. The two smaller-bore filters gave 2-11-fold improvements in signal response for representative intracellular metabolites, such as adenosine, glutamine, and leucine/isoleucine. Especially, the smallest-bore filter enabled successful detection of the metabolites in the nucleus, including tetrahydrobiopterin and cyclic guanosine monophosphate. The developed all-in-one system was explored to monitor the uptake of four anticancer drugs, including 5-fluorouracil, doxorubicin, gambogic acid, and paclitaxel in single cells, and further to investigate the drug uptake trends at the subcellular level. The all-in-one system integrates the merits of high-throughput single-cell screening and in-depth intracellular information profiling and is promising for high-coverage single-cell metabolome analysis to serve cell biology research and cancer research.

Single-cell metabolite analysis on a microfluidic chip

Metabolites can directly reflect and modulate cell responses and phenotypical changes by influencing energy balances, intercellular signals, and many other cellular functions throughout the lifespan of cells. Taking into account the heterogeneity of cells, single-cell metabolite analysis offers an insight into the functional process within one cell. Microfluidics as a powerful tool has attracted significant interest in the single-cell metabolite analysis field. The microfluidic platform is possible to observe, classify, and stimulate individual cells. It can also transport single-cell to subsequent analysis steps in a fast and controllable way to determine and analyze the composition and content of metabolites. The reviews of topics in microfluidics for single-cell metabolite analysis have been published in the past few years. However, most of them focused on metabolite analysis with mass spectrometry. Here, we covered the advances of microfluidic devices for single-cell metabolite analysis, with a focus on single-cell isolation and manipulation. What is more, we summarized the detection methods and applications of single-cell metabolites.

High-Throughput Single-Cell Mass Spectrometry Reveals Abnormal Lipid Metabolism in Pancreatic Ductal Adenocarcinoma.

Even populations of clonal cells are heterogeneous, which requires high‐throughput analysis methods with single‐cell sensitivity. Here, we propose a rapid, label‐free single‐cell analytical method based on active capillary dielectric barrier discharge ionization mass spectrometry, which can analyze multiple metabolites in single cells at a rate of 38 cells/minute. Multiple cell types (HEK‐293T, PANC‐1, CFPAC‐1, H6c7, HeLa and iBAs) were discriminated successfully. We found evidence for abnormal lipid metabolism in pancreatic cancer cells. We also analyzed gene expression in a cancer genome atlas dataset and found that the mRNA level of a critical enzyme of lipid synthesis (ATP citrate lyase, ACLY) was upregulated in human pancreatic ductal adenocarcinoma (PDAC). Moreover, both an ACLY chemical inhibitor and a siRNA approach targeting ACLY could suppress the viability of PDAC cells. A significant reduction in lipid content in treated cells indicates that ACLY could be a potential target for treating pancreatic cancer.

High‐Throughput Single‐Cell Mass Spectrometry Reveals Abnormal Lipid Metabolism in Pancreatic Ductal Adenocarcinoma

AbstractEven populations of clonal cells are heterogeneous, which requires high‐throughput analysis methods with single‐cell sensitivity. Here, we propose a rapid, label‐free single‐cell analytical method based on active capillary dielectric barrier discharge ionization mass spectrometry, which can analyze multiple metabolites in single cells at a rate of 38 cells/minute. Multiple cell types (HEK‐293T, PANC‐1, CFPAC‐1, H6c7, HeLa and iBAs) were discriminated successfully. We found evidence for abnormal lipid metabolism in pancreatic cancer cells. We also analyzed gene expression in a cancer genome atlas dataset and found that the mRNA level of a critical enzyme of lipid synthesis (ATP citrate lyase, ACLY) was upregulated in human pancreatic ductal adenocarcinoma (PDAC). Moreover, both an ACLY chemical inhibitor and a siRNA approach targeting ACLY could suppress the viability of PDAC cells. A significant reduction in lipid content in treated cells indicates that ACLY could be a potential target for treating pancreatic cancer.

Single Cell Metabolite Detection Using Inertial Microfluidics-Assisted Ion Mobility Mass Spectrometry.

Single-cell metabolite measurement remains highly challenging due to difficulties related to single cell isolation, metabolite detection, and identification of low levels of metabolites. Here, as a first step of the technological development, we propose a novel strategy integrating spiral inertial microfluidics and ion mobility mass spectrometry (IM-MS) for single-cell metabolite detection and identification. Cells in methanol suspension are inertially focused into a single stream in the spiral microchannel. This stream of separated cells is delivered to the nanoelectrospray needle to be lysed and ionized and subsequently analyzed in real time by IM-MS. This analytical system enables six to eight single-cell metabolic fingerprints to be collected per minute, including gas-phase collisional cross section (CCS) measurements as an additional molecular descriptor, giving increased confidence in metabolite identification. As a proof of concept, the metabolic profiles of three types of cancer cells (U2OS, HepG2, and HepG2.215) were successfully screened, and 19 distinct lipids species were identified with CCS value filtering. Furthermore, principal component analysis (PCA) showed differentiation of the three cancer cell lines, mainly due to cellular surface phospholipids. Taken together, our technology platform offers a simple and efficient method for single-cell lipid profiling, with additional ion mobility separation of lipids significantly improving the confidence toward identification of metabolites.

Spray-Capillary-Based Capillary Electrophoresis Mass Spectrometry for Metabolite Analysis in Single Cells.

Single-cell capillary electrophoresis mass spectrometry (CE-MS) is a promising platform to analyze cellular contents and probe cell heterogeneity. However, current single-cell CE-MS methods often rely on offline microsampling processes and may demonstrate low sampling precision and accuracy. We have recently developed an electrospray-assisted device, spray-capillary, for low-volume sample extraction. With the spray-capillary, low-volume samples (pL-nL) are drawn into the sampling end of the device, which can be used directly for CE separation and online MS detection. Here, we redesigned the spray-capillary by utilizing a capillary with a <15 μm tapered tip so that it can be directly inserted into single cells for sample collection and on-capillary CE-MS analysis. We evaluated the performance of the modified spray-capillary by performing single-cell microsampling on single onion cells with varying sample injection times and direct MS analysis or online CE-MS analysis. We have demonstrated, for the first time, online sample collection and CE-MS for the analysis of single cells. This application of the modified spray-capillary device facilitates the characterization and relative quantification of hundreds of metabolites in single cells.

A unified design allows fine-tuning of biosensor parameters and application across bacterial species

In recent years, transcriptional biosensors have become valuable tools in metabolic engineering as they allow semiquantitative determination of metabolites in single cells. Although being perfectly suitable tools for high-throughput screenings, application of transcriptional biosensors is often limited by the intrinsic characteristics of the individual sensor components and their interplay. In addition, biosensors often fail to work properly in heterologous host systems due to signal saturation at low intracellular metabolite concentrations, which typically limits their use in high-level producer strains at advanced engineering stages.We here introduce a biosensor design, which allows fine-tuning of important sensor parameters and restores the sensor response in a heterologous expression host. As a key feature of our design, the regulator activity is controlled through the expression level of the respective gene by different (synthetic) constitutive promoters selected for the used expression host. In this context, we constructed biosensors responding to basic amino acids or ring-hydroxylated phenylpropanoids for applications in Corynebacterium glutamicum and Escherichia coli. Detailed characterization of these biosensors in liquid cultures and during single-cell analysis using flow cytometry showed that the presented sensor design enables customization of important biosensor parameters as well as application of these sensors in relevant heterologous hosts.

Single-Cell Mass Spectrometry Analysis of Metabolites Facilitated by Cell Electro-Migration and Electroporation.

Single-cell metabolite analysis plays an important role in biological study. While mass spectrometry is a powerful tool for identification and quantitation of metabolites, the low absolute analyte amounts in single cell and difficulty in sampling represent significant challenges in single cell analysis. In this study, we developed an effective method with a simple sampling procedure for analyzing single cells. A single cell was driven to a capillary tip through electro-migration, followed by releasing the cell contents through electroporation, into a sealed small volume (∼1.5 pL) to prevent dilution. Subsequent mass spectrometry analysis was performed directly with nanoelectrospray ionization. This method was applied for analyzing a variety of cells and monitoring the metabolic changes in response to perturbed cell culturing conditions. This method opens a new avenue for easy and rapid analysis of single cells with high sensitivity.