Probing neuropeptide signaling at the organ and cellular domains via imaging mass spectrometry

Ye, Hui; Greer, Tyler; Li, Lingjun

doi:10.1016/j.jprot.2012.03.015

Cited by 25 publications

(29 citation statements)

References 111 publications

(142 reference statements)

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“…Unlike conventional MS that seeks high coverage of the metabolome by averaging together a large number, often millions, of cells, single-cell MS technologies are purposed to characterize biomolecular events in a cell-specific manner (17)(18)(19)(20)(21). 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.…”

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confidence: 99%

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.

178

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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mentioning

confidence: 99%

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.

178

239

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“…Microprobe sampling, matrix-assisted laser desorption ionization (13,15,16), or LC-HRMS (17) have measured peptides and proteins in a discovery (untargeted) setting in single molluscan, crustacean, frog, or mammalian oocytes (18), eggs (19), and nuclei (7). Additionally, mass cytometry was used to assay 34 targeted proteins at a high, ϳ1000 cell/s, throughput during erythropoiesis (20), and this platform was recently coupled to laser-ablation to survey 32 targeted proteins between cells in the tumor environment (21).…”

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confidence: 99%

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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“…Despite their relatively simple nervous system, crustacean model organisms employ diverse neuropeptides, including over two dozen neuropeptide families and many isoforms from several peptide families for each species [3]. Complementary to tandem MS-based neuropeptide discovery and identification process, mass spectrometric imaging (MSI) technique has been utilized to map the spatial distribution of neuropeptide families and specific isoforms directly from tissue in an anatomical context [15, 16]. The spatial localization of specific neuropeptides and determination of their co-localization patterns will provide critical information to help elucidate the underlying mechanism of cell-cell communication via signaling neuropeptide molecules.…”

Section: Overviewmentioning

confidence: 99%

Mass spectrometric analysis of spatio-temporal dynamics of crustacean neuropeptides

OuYang

Liang

2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Neuropeptides represent one of the largest classes of signaling molecules used by nervous systems to regulate a wide range of physiological processes. Over the past several years, mass spectrometry (MS)-based strategies have revolutionized the discovery of neuropeptides in numerous model organisms, especially in decapod crustaceans. Here, we focus our discussion on recent advances in the use of MS-based techniques to map neuropeptides in spatial domain and monitoring their dynamic changes in temporal domain. These MS-enabled investigations provide valuable information about the distribution, secretion and potential function of neuropeptides with high molecular specificity and sensitivity. In situ MS imaging and in vivo microdialysis are highlighted as key technologies for probing spatio-temporal dynamics of neuropeptides in the crustacean nervous system. This review summarizes the latest advancement in MS-based methodologies for neuropeptide analysis including typical workflow and sample preparation strategies as well as major neuropeptide families discovered in decapod crustaceans.

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Probing neuropeptide signaling at the organ and cellular domains via imaging mass spectrometry

Cited by 25 publications

References 111 publications

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

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)

Mass spectrometric analysis of spatio-temporal dynamics of crustacean neuropeptides

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