Single-cell Capillary Electrophoresis Research Articles

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.

Trace, Machine Learning of Signal Images for Trace-Sensitive Mass Spectrometry: A Case Study from Single-Cell Metabolomics.

Recent developments in high-resolution mass spectrometry (HRMS) technology enabled ultrasensitive detection of proteins, peptides, and metabolites in limited amounts of samples, even single cells. However, extraction of trace-abundance signals from complex data sets ( m/ z value, separation time, signal abundance) that result from ultrasensitive studies requires improved data processing algorithms. To bridge this gap, we here developed "Trace", a software framework that incorporates machine learning (ML) to automate feature selection and optimization for the extraction of trace-level signals from HRMS data. The method was validated using primary (raw) and manually curated data sets from single-cell metabolomic studies of the South African clawed frog ( Xenopus laevis) embryo using capillary electrophoresis electrospray ionization HRMS. We demonstrated that Trace combines sensitivity, accuracy, and robustness with high data processing throughput to recognize signals, including those previously identified as metabolites in single-cell capillary electrophoresis HRMS measurements that we conducted over several months. These performance metrics combined with a compatibility with MS data in open-source (mzML) format make Trace an attractive software resource to facilitate data analysis for studies employing ultrasensitive high-resolution MS.

An Integrated Chemical Cytometry Method: Shining a Light on Akt Activity in Single Cells.

Tools to evaluate oncogenic kinase activity in small clinical samples have the power to guide precision medicine in oncology. Existing platforms have demonstrated impressive insights into the activity of protein kinases, but these technologies are unsuitable for the study of kinase behavior in large numbers of primary human cells. To address these limitations, we developed an integrated analysis system that utilizes a light-programmable, cell-permeable reporter deliverable simultaneously to many cells. The reporter's ability to act as a substrate for Akt, a key oncogenic kinase, was masked by a 2-4,5-dimethoxy 2-nitrobenzyl (DMNB) moiety. Upon exposure to ultraviolet light and release of the masking moiety, the substrate sequence enabled programmable reaction times within the cell cytoplasm. When coupled to automated single-cell capillary electrophoresis, statistically significant numbers of primary human cells were readily evaluated for Akt activity.

High-Sensitivity Mass Spectrometry for Probing Gene Translation in Single Embryonic Cells in the Early Frog (Xenopus) Embryo.

Direct measurement of protein expression with single-cell resolution promises to deepen the understanding of the basic molecular processes during normal and impaired development. High-resolution mass spectrometry provides detailed coverage of the proteomic composition of large numbers of cells. Here we discuss recent mass spectrometry developments based on single-cell capillary electrophoresis that extend discovery proteomics to sufficient sensitivity to enable the measurement of proteins in single cells. The single-cell mass spectrometry system is used to detect a large number of proteins in single embryonic cells in the 16-cell embryo of the South African clawed frog (Xenopus laevis) that give rise to distinct tissue types. Single-cell measurements of protein expression provide complementary information on gene transcription during early development of the vertebrate embryo, raising a potential to understand how differential gene expression coordinates normal cell heterogeneity during development.

Mass Spec Single-Cell Proteomic Analysis

Most proteomic analyses are averages of many cells. But as the sensitivity of mass spectrometry improves, comprehensive analysis of individual cells is becoming possible. By combining single-cell capillary electrophoresis, microflow electrospray ionization, and high-resolution mass spectrometry, Peter Nemes, Sally A. Moody, and Camille Lombard-Banek of George Washington University have performed proteomic analyses of individual cells dissected from a 16-cell frog embryo (Angew. Chem. Int. Ed. 2016, DOI: 10.1002/anie.201510411). The researchers extracted and identified a total of 1,709 different proteins from three types of cells destined to develop into different parts of the frog’s body. To quantify proteins with high sensitivity, they used different mass tags to label each cell’s proteome, allowing them to detect even trace-level proteins at a low nanomolar concentration. Nearly a quarter of the proteins were common to all three cell types, but each cell type also had several hundred proteins unique to ...

Qualitative and quantitative metabolomic investigation of single neurons by capillary electrophoresis electrospray ionization mass spectrometry.

Single-cell mass spectrometry (MS) empowers metabolomic investigations by decreasing analytical dimensions to the size of individual cells and subcellular structures. We describe a protocol for investigating and quantifying metabolites in individual isolated neurons using single-cell capillary electrophoresis (CE) coupled to electrospray ionization (ESI) time-of-flight (TOF) MS. The protocol requires ∼2 h for sample preparation, neuron isolation and metabolite extraction, and 1 h for metabolic measurement. We used the approach to detect more than 300 distinct compounds in the mass range of typical metabolites in various individual neurons (25-500 μm in diameter) isolated from the sea slug (Aplysia californica) central and rat (Rattus norvegicus) peripheral nervous systems. We found that a subset of identified compounds was sufficient to reveal metabolic differences among freshly isolated neurons of different types and changes in the metabolite profiles of cultured neurons. The protocol can be applied to the characterization of the metabolome in a variety of smaller cells and/or subcellular domains.

Single-Cell Metabolomics: Changes in the Metabolome of Freshly Isolated and Cultured Neurons

Metabolites are involved in a diverse range of intracellular processes, including a cell’s response to a changing extracellular environment. Using single-cell capillary electrophoresis coupled to electrospray ionization mass spectrometry, we investigated how placing individual identified neurons in culture affects their metabolic profile. First, glycerol-based cell stabilization was evaluated using metacerebral neurons from Aplysia californica; the measurement error was reduced from ∼24% relative standard deviation to ∼6% for glycerol-stabilized cells compared to those isolated without glycerol stabilization. In order to determine the changes induced by culturing, 14 freshly isolated and 11 overnight-cultured neurons of two metabolically distinct cell types from A. californica, the B1 and B2 buccal neurons, were characterized. Of the more than 300 distinctive cell-related signals detected, 35 compounds were selected for their known biological roles and compared among each measured cell. Unsupervised multivariate and statistical analysis revealed robust metabolic differences between these two identified neuron types. We then compared the changes induced by overnight culturing; metabolite concentrations were distinct for 26 compounds in the cultured B1 cells. In contrast, culturing had less influence on the metabolic profile of the B2 neurons, with only five compounds changing significantly. As a result of these culturing-induced changes, the metabolic composition of the B1 neurons became indistinguishable from the cultured B2 cells. This observation suggests that the two cell types differentially regulate their in vivo or in vitro metabolomes in response to a changing environment.

Metabolic differentiation of neuronal phenotypes by single-cell capillary electrophoresis-electrospray ionization-mass spectrometry.

Single-cell mass spectrometry (MS) is a rapidly emerging field in metabolic investigations. The inherent chemical complexity of most biological samples poses analytical challenges when using MS platforms to measure sample content without prior chemical separation. Here, a single-cell capillary electrophoresis (CE) system was coupled with electrospray ionization (ESI) MS to enable the simultaneous measurement of a vast array of endogenous compounds in over 50 identified and isolated large neurons from the Aplysia californica central nervous system. More than 300 distinct ion signals (m/z values) were detected from a single neuron in the positive ion mode, 140 of which were selected for chemometric data analysis. Metabolic features were evaluated among six different neuron types (B1, B2, left pleural 1 (LPl1), metacerebral cell (MCC), R2, and R15) chosen for their various physiological functions. The results indicated chemical similarities among some neuron types (B1 to B2 and LPl1 to R2) and distinctive features for others (MCC and R15 cells). The quantitative nature of the MS platform allowed the comparison of metabolite levels for specific neurons. The CE-ESI-MS approach for examination of individual nanoliter-volume cells as described herein is readily adaptable to other volume-limited samples.

Reactive oxygen radical scavengers—Saving the cells?

This wet and cold season is the ideal time to develop a cold or influenza and to relieve symptoms with a mucolytic agent. N-acetylcysteine, also known as acetylcysteine or N-acetyl-L-cysteine (NAC), is a pharmaceutical drug used mainly as a mucolytic agent. Its chemical name is (R)-2-acetamido-3-sulfanylpropanoic acid. NAC is widely used as a mucolytic agent although its efficacy is highly disputed. Several other potential fields of therapeutic application such as chronic obstructive pulmonary disease, AIDS, and influenza are under discussion. NAC was also suggested as an antidote in cases of metallic intoxication and radioactive irradiation. The multitude of potentially beneficial effects are possibly related to the apoptosis alternating activities of this reactive oxygen species scavenger. However, the management of paracetamol (acetaminophen) overdose is among the most prominent therapeutical applications. Now, Löhrke and colleagues (this issue) report that NAC affects the survival of luteal cells. The authors found that cell survival markedly decreases following NAC treatment in mature corpus luteum-derived luteal cells but not in luteal cells from the developing gland. Phagocytosis and pinocytosis are microscopically the most obvious surface activities of amoebae and are morphologically and functionally similar to that in human macrophages. Antibody cross-recognition with highly conserved leukocyte cluster of differentiation (CD) marker proteins is a common finding between closely related vertebrate species. Studies on the mechanisms of phagocytosis (Ravine et al.; this issue) show similarly recognized protein epitopes on the cell surface. For example, the human panleukocytic marker, CD45 or the protein tyrosine phosphatase receptor type C are important regulators of human leukocyte activity being highly expressed in all nucleated hematopoietic cell lines at all maturation stages. Phagocytosis in the phylogenesis is judged to be a highly conservative mechanism due to the cross-reactivity of different antibodies against the human CD45 antigen with antigen epitopes of amoebae. Proximity of molecules is of substantial relevance in understanding molecular interactions and signal transduction pathways. Typical fields of application are T-cell receptor analysis or Her2 hetero- or homo-dimerization in cancer (Vollmann-Zwerenz and colleagues; this issue). Förster resonance energy transfer is nowadays a convenient method for flow and image cytometry to quantitate molecule proximity. An elegant new alternative is the recently developed method of fluorescence proximity ligation (1). Bridgeman and collaborators (this issue) present a completely new approach to determine molecular interactions in making use of the old co-precipitation technology used for decades in protein chemistry for flow cytometry. This bead-based assay can be multiplexed and renders high detection sensitivity. Determining changes in enzyme activity and protein modification on a single cell basis is of substantial importance for identifying heterogeneity of cell systems. However, present technology lacks the sensitivity to identify several single peptide fragments. The only single cell protein technology is single cell capillary electrophoresis but it lacks the ability to definitely identify protein fragments and other reaction products. In this issue, Brown and colleagues elegantly combined single cell capillary electrophoresis with liquid chromatography and mass spectrometry in a workflow. Using this stream of technologies, the authors were able to identify peaks measured by single cell capillary electrophoresis. This approach may lead to unraveling metabolic pathways and enzyme activities that are of extreme relevance in several diseases. Development of assays for the automated and unbiased quantitation of microscopic images is an ongoing process of software and algorithm research (2). The major challenges are: How can cells or cell organelles be unequivocally recognized and how can these objects then be objectively categorized? Yu et al. (this issue) propose an upgrade of their previously presented maximum common boundary (MCB) algorithm (3) that they applied to measure neurite outgrowth. The new upgrade of their algorithm is based on Voronoi diagrams. This approach improves and speeds up image segmentation of images from a neuroblastoma cell line cultured at different cell densities. Forero and colleagues (this issue) propose a highly interesting approach to automatically count neurons from Drosophila embryos. Their program (DeadEasy Neurons) will be of interest to researchers from neurobiology and developmental biology. DeadEasy Neurons is an image processing and object recognition method that identifies neuronal nuclei labeled with HB9 over background (nonspecific antibody staining) from a stack of confocal images that make up the complete embryonic ventral nerve cord, and it automatically counts the neurons in 3D. The method (written in Java) will become freely available through ImageJ with this publication, and the parameters indicated and the algorithm can be modified by the user to count cells of choice. Dr. Jozsef Bocsi, Heart Center Leipzig, is acknowledged for his help with this editorial.

Single amino acid resolution of proteolytic fragments generated in individual cells

The complex nature of enzyme regulation mandates that enzyme activity profiles be measured in the context of the intact cell. Single-cell capillary electrophoresis (CE) coupled with laser-induced fluorescence is a powerful approach for quantitation and separation of analytes present in small samples and single live cells; however, it does not allow for the definitive identification of the reaction products. On the other hand, mass spectrometry (MS) is able to identify analytes but still lacks the requisite sensitivity for most single-cell analysis applications. Thus, it follows that by determining the relative amounts of reaction products generated in single cells using CE and by producing larger quantities of these products using bulk cell populations to identify them using MS, it is possible to determine enzyme activity profiles in single cells. In this study, the applicability of this approach was demonstrated by examining the intracellular fate of a protease substrate derived from the beta-amyloid precursor protein (beta-APP). In single live TF-1 cells, three distinct fragments were generated from the beta-APP peptide, which differed by a single uncharged amino acid. The CE measurements indicated that the proteolytic fragment profiles (i.e., the relative amounts of each fragment) were consistent from cell to cell but that they were different from those obtained in cell lysates. Furthermore, measurements obtained at the single cell level made it possible to observe a modest but statistically significant negative correlation between the total amount of beta-APP peptide loaded in cells and the fraction of peptide that remained intact. This study demonstrates how single-cell CE, MS, and peptide substrates can be combined to identify and measure enzyme activities in single live cells.

Microfabricated system for parallel single-cell capillary electrophoresis.

Performing single-cell electrophoresis separations using multiple parallel microchannels offers the possibility of both increasing throughput and eliminating cross-contamination between different separations. The instrumentation for such a system requires spatial and temporal control of both single-cell selection and lysis. To address these problems, a compact platform is presented for single-cell capillary electrophoresis in parallel microchannels that combines optical tweezers for cell selection and electromechanical lysis. Calcein-labeled acute myloid leukemia (AML) cells were selected from an on-chip reservoir and transported by optical tweezers to one of four parallel microfluidic channels. Each channel entrance was manufactured by F2-laser ablation to form a 20- to 10-microm tapered lysis reservoir, creating an injector geometry effective in confining the cellular contents during mechanical shearing of the cell at the 10-microm capillary entrance. The contents of individual cells were simultaneously injected into parallel channels resulting in electrophoretic separation as recorded by laser-induced fluorescence of the labeled cellular contents.

Nuclear localization of aminoacyl-tRNA synthetases using single-cell capillary electrophoresis laser-induced fluorescence analysis.

Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes whose function in specific aminoacylation of tRNAs is central to the process of protein translation, which occurs in the cytoplasm of all living cells. In addition to their well-established cytoplasmic localization, fluorescence microscopy studies and analysis of the aminoacylation state of nuclear tRNAs have revealed that synthetases are localized in the nuclei of cells from several species including Xenopus laevis and Saccharomyces cerevisiae. Whether nuclear localization of aaRSs is a general phenomenon that occurs in all eukaryotic cells is an open question. In the work described here, human methionyl-tRNA synthetase (MRS) and human lysyl-tRNA synthetase (KRS) were expressed in human-derived DeltaH2-1 osteosarcoma cells as enhanced green fluorescent protein (EGFP) fusion proteins. The subcellular localization of these EGFP-aaRSs was first probed by fluorescence microscopy using cells that coexpressed EGFP-aaRS and a nuclear marker fusion protein, nuDsRed. As expected, both aaRSs were present in the cytosol, while only EGFP-MRS was also clearly localized in the nucleus. To confirm these findings, and to investigate a potentially more sensitive, general method for nuclear localization studies, capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze single DeltaH2-1 cells expressing both EGFP-aaRS and nuDsRed. While cytosolic EGFP signals were detected for both EGFP-MRS and EGFP-KRS, only EGFP-MRS was found in the nucleus, along with nuDsRed. The detection of EGFP-MRS in nuclei of DeltaH2-1 cells demonstrates the feasibility of using CE-LIF analysis in nuclear localization studies of proteins in mammalian cells.

Identification of proteins in single-cell capillary electrophoresis fingerprints based on comigration with standard proteins.

In the previous paper in this Journal, we reported the use of capillary sieving electrophoresis to characterize proteins expressed by single cancer cells at specific phases in the cell cycle. Analysis of the data revealed one component with cell cycle-dependent changes in expression at the 99% confidence limit. However, the amount of protein present in a single cell is far too small to allow its direct identification by mass spectrometry. In this paper, we report a method by which such proteins can be tentatively identified. We perform standard SDS-PAGE electrophoresis of the proteins contained within a homogenate prepared from an HT29 cell culture. Proteins extracted from bands in the gel are identified by mass spectrometry. The proteins also provide a set of standards that can be used to spike the sample before capillary sieving electrophoresis (CSE) separation; comigration is taken as evidence for the identity of the target protein. In a proof-of-principle experiment, a single band migrating at approximately 47 kDa was isolated from the SDS-PAGE gel generated from the HT29 cell line. Proteins extracted from this band were used to spike a CSE separation of the same extract. This band comigrated with a cell cycle-dependent component identified from single-cell analysis. In-gel digestion and LC/MS/MS were used to identify five proteins, including cytokeratin 18, which is the product of the most highly expressed gene in this cell line.

Laser-induced native fluorescence (LINF) imaging of serotonin depletion in depolarized neurons

Since certain neurotransmitters exhibit native fluorescence we can monitor this property to disclose intracellular changes that result from neurotransmitter release. Isolated Retzius neurons of the leech are known to release serotonin (5-HT) during depolarization. Using intensified CCD technology coupled with UV laser (305 nm) excitation we observed depolarization and calcium-dependent reductions in native fluorescence in the axon, as well as in the cortex of the cell body. When taken together with data obtained from single-cell capillary electrophoresis, we demonstrate that this laser-induced native fluorescence can be reliably used to study spatial and temporal changes in intracellular transmitter content that accompany calcium-dependent secretion.