Name: Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo (Video) | JoVE Journal
Uploaded: 2022-04-21T13:00:00.000+00:00
Duration: 9 min 18 s
Description: Discover mass spectrometry proteomics for developmental biology using Xenopus laevis embryos. This lineage-guided method reveals spatial and temporal protein dynamics during cell fate commitment and tissue induction.

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We are grateful to Jie Li (University of Maryland, College Park) for valuable discussions on embryonic dissociation and FACS. We thank Vi M. Quach and Camille Lombard-Banek for assistance with sample preparation and data collection in previous studies exemplifying the proteomic applications that are highlighted in this protocol. Parts of this work were supported by the National Science Foundation under award number IOS-1832968 CAREER (to P.N.), the National Institutes of Health under award number R35GM124755 (to P.N.), the University of Maryland-National Cancer Institute Partnership Program (to P.N.), and COSMOS Club Foundation research awards (to A.B.B. and L.R.P.).

The authors declare no competing interests.

This protocol enables the characterization of protein expression in identified cell lineages in embryos of the Xenopus species. Stemming from HRMS, the methodology combines exquisite specificity in molecular identification, capability for multi-protein detection without molecular probes (usually hundreds to thousands of different proteins), and a capability for quantification. Adaptability to classical tools and workflows in cell and developmental (neuro)biology expand HRMS proteomics to exciting applications, i...

Our understanding of cell differentiation and the genesis of tissues and organs is the result of decades of elaborate targeted screens of genes and their products. Increasing our knowledge of all the biomolecules and their quantities during important cellular events would help unravel molecular mechanisms that control the spatial and temporal patterning of the vertebrate body plan. Technologies enabling molecular amplification and sequencing are now able to routinely report on large numbers of genes and transcripts, supporting hypothesis-driven studies in basic biological and translational research. To understand developing systems, a complex relationship between transcription and translation advocates for direct analysis of multiple proteins and their post-translational modifications. Global proteomics using in vitro biological systems, such as induced pluripotent stem cells, began to delineate mechanisms of tissue induction1,2. In complex organisms, such as the vertebrate embryo, development relies on morphogen gradients in the context of space and time3. It follows that gaining knowledge of proteomic changes as cells differentiate to form specialized tissues, such as neural tissues, offers a key to unlock molecular programs controlling normal and defective development and guide next-generation therapeutics.
The vertebrate South African clawed frog (Xenopus laevis) is a well-established model in cell and developmental, neuro-, and regenerative biology. Sir John Gurdon's 2012 Nobel Prize in Physiology or Medicine4,5 for the discovery of pluripotency of the somatic nucleus highlighted the importance of this model for discoveries in basic and translational studies. Xenopus embryos develop externally to the mother, thus facilitating direct manipulation of cells, cell clones, and gene expression over various stages of development. Asymmetrical pigmentation and stereotypical cell divisions enabled the charting of reproducible fate maps from the 16-6 and 32-cell7,8 stage embryo. For high-resolution mass spectrometry (HRMS) based proteomics, additional advantages of the model include relatively large size (~1 mm in diameter), which yields abundant protein content for analysis (~130 µg in early cleavage-stage embryos, ~10 µg of protein content in single cells of the 16-cell embryo)9,10.
At present, HRMS is the leading technology of choice for detecting proteins. This technology enables direct, sensitive, and specific detection and quantification of multiple, usually hundreds-to-thousands of different proteins11. Bottom-up proteomics by HRMS involves a series of interconnected steps. Following extraction from the cell/tissue sample, proteins are digested with a proteolytic enzyme, such as trypsin (bottom-up proteomics). The resulting peptides are separated based on their different physicochemical properties, including hydrophobicity (reversed-phase liquid chromatography, LC), net charge (ion-exchange chromatography), size (size exclusion chromatography), or electrophoretic mobility (capillary electrophoresis, CE). Peptides are then charged (ionized), typically using electrospray ionization (ESI), and peptide ions are detected and sequenced via gas-phase fragmentation by tandem HRMS. The resulting peptide data are mapped to the proteome of the organism being studied. With protein-specific (proteotypic) peptide ion signal intensity correlating with concentration, protein quantification can be performed label-free or label-based (multiplexing quantitation). HRMS proteomics yields a rich resource of information on the molecular state of the system under study, allowing for the generation of hypotheses and follow-up functional studies.
<img alt="Cell microaspiration and isolation diagram; embryo to gastrula, sorting via FACS, experimental setup." class="xfigimg" src="/files/ftp_upload/63586/63586fig01v2.jpg" data-altupdatedat="1788282455000" data-alt="Figure 1"> 
Figure 1: Spatiotemporally scalable proteomics enabling cell-lineage guided HRMS proteomics in the developing (frog) embryo. (A) Visualization of the specimen (1) using a stereomicroscope (2) for injection of an identified cell (inset), using a fabricated micropipette (3) under control by a translation-stage (4). (B) Subcellular sampling of the identified left D11 cell in a 16-cell embryo. (C) Dissection of a whole D11 cell from a 16-cell embryo. (D) Fluorescent (green) tracing of the left and right D111 progenies from a 32-cell embryo to guide dissection of the neural ectoderm (NE) in the gastrula (stage 10) and isolation of the descendent tissue from the tadpole using FACS. Scale bars: 200 µm for embryos, 1.25 mm for the vial. Figures were adapted with permission from references15,19,21,59. <a href="https://www.jove.com/files/ftp_upload/63586/63586fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The protocol presented here enables HRMS-based quantification of large numbers of proteins in identified cells/tissues in developing X. laevis embryos. The approach builds on accurate cell identification, reproducible cell fate maps, and established methodologies to track cell lineages in this biological model6,7,8. As shown in Figure 1, we study proteomes from single cells by employing whole-cell dissection or capillary microsampling to aspirate cellular content. Monitoring the lineage of a cell permits us to study the spatiotemporal evolution of the proteome as cells form tissues during gastrulation. The cell progeny is fluorescently marked by injecting a fluorophore conjugated to inert dextran or mRNA for fluorescent protein (e.g., green fluorescent protein, or GFP). The labeled progeny is isolated at desired developmental time points. During gastrulation, cell clones that are tightly clustered may be isolated by dissection. After gastrulation, cell clones may be distributed within the embryo owing to migratory movements and can be isolated from dissociated tissues by fluorescence-activated cell sorting (FACS). Proteins in these cells and tissues are measured via bottom-up proteomics employing HPLC or CE for separation and ESI tandem HRMS for identification. Cell-lineage-guided HRMS proteomics is scalable to different cell sizes and lineages within the embryo and is specific, sensitive, and quantitative. Through select examples shown here, we also demonstrate this protocol to be scalable and broadly adaptable to different types of cells and cell lineages.
<img alt="Cell and tissue dissection workflow diagram with peptide digestion, chromatography, ESI-HRMS analysis." class="xfigimg" src="/files/ftp_upload/63586/63586fig02.jpg" data-altupdatedat="1788282455000" data-alt="Figure 2"> 
Figure 2: The bioanalytical workflow. Micro-dissection and capillary aspiration, or FACS facilitated sampling of cellular and clonal protein content. Depletion of abundant yolk proteins and separation by capillary electrophoresis (CE) or nano-flow liquid chromatography (LC) enhanced identification (ID) sensitivity using electrospray ionization (ESI) high-resolution mass spectrometry (HRMS). Quantification revealed dysregulation, supplying new information for hypothesis-driven studies in conjunction with information available from gene ontology (GO). Figures were adapted with permission from reference15. <a href="https://www.jove.com/files/ftp_upload/63586/63586fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Acetonitrile (LC-MS-grade)</td><td>Fisher Scientific</td><td>A955</td><td></td></tr><tr><td>Agarose</td><td>ThermoFisher Scientific</td><td>R0492</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Fisher Scientific</td><td>A643-500</td><td></td></tr><tr><td>Analytical Column</td><td>Thermo Scientific</td><td>164941</td><td></td></tr><tr><td>Analytical microbalance</td><td>Mettler-Toledo</td><td>XSE105DU</td><td></td></tr><tr><td>Automatic peptide fractionation platform</td><td>Agilent</td><td>1260 Infinity II</td><td></td></tr><tr><td>Borosilicate Capillaries</td><td>Sutter Instruments Co.</td><td>B100-50-10</td><td></td></tr><tr><td>Borosilicate Capillaries (for making Emmitters)</td><td>Sutter Instruments</td><td>B100-75-10</td><td></td></tr><tr><td>C18 spin columns (for desalting)</td><td>ThermoFisher Scientific</td><td>89870</td><td></td></tr><tr><td>Camera ro monitor electrospray</td><td>Edmund Optics Inc.</td><td>EO-2018C</td><td></td></tr><tr><td>Combretastatin A4</td><td>Millipore Sigma</td><td>C7744</td><td></td></tr><tr><td>Commercial CESI system</td><td>AB SCIEX</td><td>CESI</td><td></td></tr><tr><td>(Cyclohexylamino)-1-propanesulfonic acid (CAPS)</td><td>VWR</td><td>97061-492</td><td></td></tr><tr><td>Cytochalasin D</td><td>Millipore Sigma</td><td>C8273</td><td></td></tr><tr><td>Dextran, Alexa Fluor 488; 10,000 MW, Anionic, Fixable</td><td>ThermoFisher Scientific</td><td>D22910</td><td></td></tr><tr><td>Diothiothreitol</td><td>Fisher Scientific</td><td>FERR0861</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11252-30</td><td></td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>AAJ62786AP</td><td></td></tr><tr><td>Epifluorescence light source</td><td>Lumencore</td><td>AURA III</td><td></td></tr><tr><td>Eppendorf LoBing microcentrifuge&amp;nbsp; tubes: protein</td><td>Fisher Scientific</td><td>13-698-793</td><td></td></tr><tr><td>Formic acid (LC-MS-grade)</td><td>Fisher Scientific</td><td>A117-50</td><td></td></tr><tr><td>Freezer (-20 &amp;deg;C)</td><td>Fisher Scientific</td><td>97-926-1&amp;nbsp;</td><td></td></tr><tr><td>Freezer (-80 &amp;deg;C)</td><td>Thermo Scientific</td><td>TSX40086A</td><td></td></tr><tr><td>Fused silica capillary</td><td>Molex</td><td>1088150596</td><td></td></tr><tr><td>Heat Block</td><td>Benchmark</td><td>BSH300</td><td></td></tr><tr><td>High pressure liquid Chromatography System</td><td>ThermoFisher Scientific</td><td>Dionex Ultimate 3000 RSLC nanosystem</td><td></td></tr><tr><td>High voltage power supply</td><td>Spellman</td><td>CZE1000R</td><td></td></tr><tr><td>High-resolution Mass Spectrometer</td><td>ThermoFisher Scientific</td><td>Orbitrap Fusion Lumos Tribrid Mass Spectrometer</td><td></td></tr><tr><td>HPLC caps</td><td>Thermo Scientific</td><td>C4013-40A</td><td></td></tr><tr><td>HPLC Vials</td><td>Thermo Scientific</td><td>C4013-11</td><td></td></tr><tr><td>Illuminator e.g. Goosenecks</td><td>Nikon</td><td>C-FLED2</td><td></td></tr><tr><td>Ingenuity Pathway Analysis</td><td>Qiagen</td><td></td><td></td></tr><tr><td>Iodoacetamide</td><td>Fisher Scientific</td><td>AC122275000</td><td></td></tr><tr><td>Methanol (LC-MS-grade)</td><td>Fisher Scientific</td><td>A456</td><td></td></tr><tr><td>Methanol (LC-MS-grade)</td><td>Fisher Scientific</td><td>A456-4</td><td></td></tr><tr><td>Microcapillary puller</td><td>Suttor Instruments</td><td>P-2000</td><td></td></tr><tr><td>Microinjector</td><td>Warner Instrument, Handem, CT</td><td>PLI-100A</td><td></td></tr><tr><td>Micropippette puller</td><td>Sutter Instruments Co.</td><td>P-1000</td><td></td></tr><tr><td>MS data analysis software, commercial</td><td>ProteomeDiscoverer</td><td></td><td></td></tr><tr><td>MS data analysis software, opensource</td><td>MaxQuant</td><td></td><td></td></tr><tr><td>non-idet 40 substitute</td><td>Millipore Sigma</td><td>11754599001</td><td></td></tr><tr><td>Petri dish 60 mm and 80 mm</td><td>Fisher Scientific</td><td>S08184</td><td></td></tr><tr><td>Pierce 10 &amp;micro;L bed Zip-tips (for desalting)</td><td>ThermoFisher Scientific</td><td>87782</td><td></td></tr><tr><td>Pierce bicinchoninic acid protein assay kit</td><td>ThermoFisher Scientific</td><td>23225</td><td></td></tr><tr><td>Pierce quantitative colorimetric peptide assay</td><td>ThermoFisher Scientific</td><td>23275</td><td></td></tr><tr><td>Pierce Trypsin Protease (MS Grade)</td><td>Fisher Scientific</td><td>PI90058</td><td></td></tr><tr><td>Protein LoBind vials</td><td>Eppendorf</td><td>0030108434&lt;br/&gt; , 0030108442</td><td></td></tr><tr><td>Refrigerated Centrifuge</td><td>Eppendorf</td><td>5430R</td><td></td></tr><tr><td>Refrigerated Incubator</td><td>Thermo Scientific</td><td>PR505755R/3721</td><td></td></tr><tr><td>sodium isethionate</td><td>Millipore Sigma</td><td>220078</td><td></td></tr><tr><td>sodium pyrophosphate</td><td>Sigma Aldrich</td><td>221368-100G</td><td></td></tr><tr><td>Stainless steel BGE vial</td><td>Custom-Built</td><td></td><td></td></tr><tr><td>Stainless steel sample vials</td><td>Custom-Built</td><td></td><td></td></tr><tr><td>Stereomicroscope (objective 10x)</td><td>Nikon</td><td>SMZ 1270, SZX18</td><td></td></tr><tr><td>Sucrose</td><td>VWR</td><td>97063-790</td><td></td></tr><tr><td>Syringe pumps (2)</td><td>Harvard Apparatus</td><td>704506</td><td></td></tr><tr><td>Syringes (gas-tight): 500&amp;ndash;1000 &amp;micro;L</td><td>Hamilton</td><td>1750TTL</td><td></td></tr><tr><td>Transfer pipettes (Plastic, disposable)</td><td>Fisher Scientific</td><td>13-711-7M</td><td></td></tr><tr><td>Trap Column</td><td>Thermo Scientific</td><td>164750</td><td></td></tr><tr><td>Tris-HCl (1 M solution)</td><td>Fisher Scientific</td><td>AAJ22638AP</td><td></td></tr><tr><td>Vacuum concentrator capable of operation at 4&amp;ndash;10 &amp;deg;C</td><td>Labconco</td><td>7310022</td><td></td></tr><tr><td>Vortex-mixer</td><td>Benchmark</td><td>BS-VM-1000</td><td></td></tr><tr><td>Water (LC-MS-grade)</td><td>Fisher Scientific</td><td>W6</td><td></td></tr><tr><td>Water (LC-MS-grade)</td><td>Fisher Scientific</td><td>W6</td><td></td></tr><tr><td>XYZ translation stage</td><td>Thorlabs</td><td>PT3</td><td></td></tr><tr><td>XYZ translation stage</td><td>Custom-Built</td><td></td><td></td></tr></tbody></table>

cell-lineage guided mass spectrometry proteomics in the developing (frog) embryo

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

This protocol enabled the study of proteins in single cells and their lineages as they establish tissues in X. laevis embryos. Figure 1 illustrates one such application of the approach to study proteins in neural-tissue-fated cells and the newly induced neural ectoderm in the embryo. As shown in Figure 1A, the bioanalytical workflow integrated traditional tools of cell and developmental biology to identify, inject/aspirate cells, and collect specimens. ...

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

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Research

JoVE Journal

Chemistry

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq—the most popular approach to investigate chromatin features at a global level—is outlined here for Xenopus embryos.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Light sheet-based fluorescence microscopy is the most valuable tool in developmental biology. A major issue in comparative studies is ambient variance. Our protocol describes an experimental framework for simultaneous live imaging of multiple specimens and, therefore, addresses this issue pro-actively.

Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes

Zebrafish targeting reactive electrophiles and oxidants (Z-REX) is a chemical biology-based method for the investigation of reactive small-molecule signaling. This technique can be applied to live fish of different developmental stages. Here, we couple standard assays in zebrafish with Z-REX for signaling pathway analysis.

Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites

Presented here is an effective rapid blood perfusion protocol to prepare tissue samples from African clawed frogs for transcriptomics and proteomics studies.

Effective Rapid Blood Perfusion in Xenopus

Discover mass spectrometry proteomics for developmental biology using Xenopus laevis embryos. This lineage-guided method reveals spatial and temporal protein dynamics during cell fate commitment and tissue induction.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

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