Analysis of Single Cells with Capillary Electrophoresis Electrospray Ionization Fourier Transform Ion Cycloton Resonance Mass Spectrometry

Hofstadler, Steven A.; Severs, Joanne C.; Smith, Richard D.; Swanek, Franklin D.; Ewing, Andrew G.

doi:10.1002/(sici)1097-0231(19960610)10:8<919::aid-rcm597>3.0.co;2-8

Cited by 126 publications

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“…1 Numerous analytical techniques, including electrochemical detection, 2-6 separation-based methods, 7-9 fluorescence microscopy, 10-13 and mass spectrometry [14][15][16][17][18][19][20][21][22][23][24] have been used to surmount the inherent difficulties of measurements down to the μm-, pL-, and zmole-scale to study the intricate chemistry that exists within single cells.…”

Section: Introductionmentioning

confidence: 99%

Secondary Ion MS Imaging To Relatively Quantify Cholesterol in the Membranes of Individual Cells from Differentially Treated Populations

et al. 2007

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Time of flight secondary ion mass spectrometry (ToF-SIMS) is a well-established bioanalytical method for directly imaging the chemical distribution across single-cells. Here we report a protocol for the use of SIMS imaging to comparatively quantify the relative difference in lipid composition between the plasma membranes of two cells. This development enables direct comparison of the chemical effects of different drug treatments and incubation conditions in the plasma membrane at the single-cell level. Relative, quantitative ToF-SIMS imaging has been used here to compare macrophage cells treated to contain elevated levels of cholesterol with respect to control cells. Insitu fluorescence microscopy with two different membrane dyes was used to discriminate morphologically similar but differentially treated cells prior to SIMS analysis. SIMS images of fluorescently identified cells reveal that the two populations of cells have distinct outer leaflet membrane compositions with the membranes of the cholesterol-treated macrophages containing more than twice the amount of cholesterol of control macrophages. Relative quantification with SIMS to compare the chemical composition of single-cells can provide valuable information about normal biological functions, causative agents of diseases, and possible therapies for diseases.

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Section: Introductionmentioning

confidence: 99%

Secondary Ion MS Imaging To Relatively Quantify Cholesterol in the Membranes of Individual Cells from Differentially Treated Populations

et al. 2007

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“…For example, targeted experiments by microarrays for MS recently probed the metabolic mechanism of perturbation in yeast cells that were masked by traditional population-averaging approaches (22), and atmospheric-pressure laser desorption/ablation (13,23) and direct microsampling electrospray ionization (ESI) (24,25) have also found differences between single cells. Encouraged by the earlier success of capillary electrophoresis (CE) MS in the proteomic analysis of single erythrocytes (26)(27)(28), we have recently extended single-cell microdissection and CE-microflow ESI (μESI) MS to small molecules to broaden the coverage of the metabolome via chemical separation (29). By removing detection interferences, single-cell CE-μESI-MS was able to detect various small molecules including neurotransmitters in single neurons of the Aplysia californica.…”

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Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo

Onjiko

Moody

Nemes

2015

Proc. Natl. Acad. Sci. U.S.A.

174

239

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Spatial and temporal changes in molecular expression are essential to embryonic development, and their characterization is critical to understand mechanisms by which cells acquire different phenotypes. Although technological advances have made it possible to quantify expression of large molecules during embryogenesis, little information is available on metabolites, the ultimate indicator of physiological activity of the cell. Here, we demonstrate that single-cell capillary electrophoresis-electrospray ionization mass spectrometry is able to test whether differential expression of the genome translates to the domain of metabolites between single embryonic cells. Dissection of three different cell types with distinct tissue fates from 16-cell embryos of the South African clawed frog (Xenopus laevis) and microextraction of their metabolomes enabled the identification of 40 metabolites that anchored interconnected central metabolic networks. Relative quantitation revealed that several metabolites were differentially active between the cell types in the wild-type, unperturbed embryos. Altering postfertilization cytoplasmic movements that perturb dorsal development confirmed that these three cells have characteristic small-molecular activity already at cleavage stages as a result of cell type and not differences in pigmentation, yolk content, cell size, or position in the embryo. Changing the metabolite concentration caused changes in cell movements at gastrulation that also altered the tissue fates of these cells, demonstrating that the metabolome affects cell phenotypes in the embryo.single cell | mass spectrometry | metabolomics | embryo development | Xenopus T he ability to understand the basic mechanisms that regulate embryonic development requires knowledge of the complete suite of biomolecules expressed by each cell in the organism. Although technological advances in single-cell isolation, genome sequencing, and transcriptome analyses have made it possible to determine spatial and temporal changes for large molecules (1, 2), little is known about small-molecular events that unfold in the individual cells (blastomeres) of the embryo. In many animals, mRNAs and proteins synthesized during oogenesis are sequestered to different cytoplasmic domains, which after fertilization then specify the germ-cell lineage and determine the anteriorposterior and dorsal-ventral axes of the embryo. For example, in the South African clawed frog (Xenopus laevis), several mRNAs are localized to the animal pole region (Fig. 1), which later gives rise to the embryonic ectoderm and the nervous system (3), whereas VegT mRNA localization to the vegetal pole specifies endoderm formation (4), and region-specific relocalization of the Wnt and Dsh maternal proteins govern the dorsal-ventral patterning of the embryo (5). However, there is abundant evidence that in developing systems not all transcripts are translated into proteins, and not all proteins are active; therefore, analyses of the mRNAs and proteins within single cells may not reveal...

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“…Electrophoretic separation affords exquisite peak capacity, is compatible with diverse types of molecules, can be hyphenated to ESI-HRMS via various interface designs, and is scalable to single cells (22)(23)(24)(25)(26)(27). Using microscale chemical separation to simplify sample chemistry, capillary electrophoresis (CE), and Fourier transform MS was used to target ␣-and ␤-globulins in 5-10 human erythrocytes (28) and carbonic anhydrase (23) in lysates diluted to single cells. A microfluidic setting extended these electrophoretic studies to higher throughput, 12 erythrocytes/min (24).…”

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Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS)

Lombard‐Banek¹,

Reddy²,

Moody

et al. 2016

Molecular & Cellular Proteomics

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Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ϳ75-amol (ϳ11 nM) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or <0.2% of the total protein contained in a blastomere in the 16-cell embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n ‫؍‬ 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level. Molecular & Cellular

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Analysis of Single Cells with Capillary Electrophoresis Electrospray Ionization Fourier Transform Ion Cycloton Resonance Mass Spectrometry

Cited by 126 publications

References 0 publications

Secondary Ion MS Imaging To Relatively Quantify Cholesterol in the Membranes of Individual Cells from Differentially Treated Populations

Secondary Ion MS Imaging To Relatively Quantify Cholesterol in the Membranes of Individual Cells from Differentially Treated Populations

Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo

Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS)

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