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

This lineage guided approach enables proteomic analysis of important developmental events with spatial and temporal resolution within the vertebrate embryo imparting information that would not be accessible through whole embryo measurements. This approach can be readily extended to study proteins, metabolites and transcripts from different embryonic cells as they differentiate during development. This method can advance our understanding of molecular mechanisms that control normal and disease development, including mechanisms of tissue induction and cell fate commitment during early development.

Use a hair loop to guide each embryo into a well and gently position them so that the targeted cell of interest is at the right angle to the microneedle. Follow the xenopus laevis tissue fate maps to identify the precursor cell of the lineage of interest. Use two sharpen forceps to gently remove the vitelline membrane surrounding the embryo.

Isolate the single cells from the embryo by manual dissection using forceps. Begin by setting up the injection needle containing the lineage tracer solution. Mount the microinjection needle into a micro pipette holder controlled by a multi-axis micro manipulator.

Connect the micro pipette holder to a micro injector and fill the needle with the lineage tracer by applying negative pressure. Calibrate the needle and adjust the size of the needle tip and injection time to deliver approximately one nanoliter of the lineage tracer solution measured in mineral oil. Ensure that the droplet size being injected is accurate and adjust the injector settings if required.

Flood the microinjection clay dish with a 3%fCAL solution and transfer approximately 10 embryos to the clay dish using a transfer pipette. Inject the cells of interest with approximately one nanoliter of the fluorescent dextran or 200 picograms of GFP mRNA. Using a stereo microscope, confirm the success of cell labeling.

Ensure that only the intended cell is injected. Discard embryos containing injured or incorrectly labeled cells following institutional policies. Transfer the injected embryos to a petri dish containing 0.5x Steinberg solution.

Culture them between 14 to 25 degrees Celsius until they reach the desired developmental stage. Transfer three to five embryos to an agar dish containing 0.2x Steinberg solution for micro dissections. Use forceps to dissect the labeled clone from the embryo.

Collect the dissected tissue with a 0.5 to 10 microliter pipette and deposit them into a micro centrifuge file. Using a pipette, aspirate the media surrounding the collected tissue to limit salts in the sample to avoid interference with HRMS analysis. Immediately freeze the isolated cells by placing the sample vial on dry ice or liquid nitrogen.

Store the samples at minus 80 degrees Celsius until mass spectrometry analysis. For single cell sample processing by CE, denature the proteins by heating the sample to 60 degrees Celsius for approximately 15 minutes. Then equilibrate the sample to room temperature for five minutes and add trypsin to the samples for digestion.

For analysis by NanoLC, lyse up to five dissected tissues in 50 microliters of lysis buffer. Facilitate the process by pipetting the sample up and down. To process the dissected tissues, incubate the lysate at four degrees Celsius for 10 minutes.

Then pellet the cell debris and yolk platelets at four degrees Celsius by centrification at 4, 500 RCF. Transfer the supernatant into a clean micro centrifuge vial and add 10%SDS to obtain a final concentration of 1%SDS in the lysate. For tissues, add 0.5 molar dithiothreitol to the lysate to obtain a final concentration of approximately 25 millimolar.

Then incubate the lysate for 30 minutes at 60 degrees Celsius to chemically reduce disulfide bonds in proteins. Add 0.5 molar iodoacetamide to obtain the final concentration of approximately 75 millimolar in the lysate and incubate the mixture for 15 minutes at room temperature in the dark. Add 0.5 molar dithiothreitol same as the initial volume to quench the reactants remaining from the alkylation reaction.

Precipitate proteins in acetone overnight as described in the text protocol and reconstitute in 0.5 molar ammonium bicarbonate. Add trypsin for sample digestion and incubate the vials at 37 degrees Celsius. To separate the peptides using CE, reconstitute the protein digested in one to two microliters of the sample solvent.

Vortex the mixed sample and centrifusion at 10, 000 RCF for two minutes at four degrees Celsius to pellet cell debris. Initialize the CE-ESI instrument by flushing the CE capillary with the BGE. Place one microliter of sample into the sample vial and inject one to 10 nanoliters of the sample into CE separation capillary by hydrodynamic injection.

Transfer the inlet end of the CE separation capillary into the BGE. Start electrophoretic separation by gradually ramping the CE separation voltage from earth ground. Potentials of 20 to 28 kilovolts with a current below 10 micro amperes ensure stable and reproducible instrumental performance for analysis.

To separate using Nano LC, re-suspend the peptide sample in mobile phase A.The sample's concentration and injection volume depend on the available LC system and column. Transfer the sample into an LC vial. Load approximately 200 nanograms to two micrograms of peptide sample into the C18 analytical column and separate the peptides at a flow rate of 300 nanoliters per minute using gradient elution as described in the text manuscript.

Using a camera, check the liquid flow through the electro spray emitter and visually inspect the setup for possible leaks. Acquire mass spectrometry events and sequence the peptides as described in the text manuscript. Gene translational differences were detected between D11 cells dissected from different embryos using CE-ESI-HRMS.

Lineage-labeled dissected cell clones were analyzed by LC-HRMS. Pathway analysis of proteins showed upregulated protein translation and energy metabolism in the Spemann's organizer with the neuroectoderm. The neuroectoderm prodeum was enriched in proteins associated with nuclear transport of protein cargo in the cell likely indicating downstream events following signaling.

The enrichment analysis of molecular functions indicated upregulation in translation initiation, RNA binding and binding of the proteasome complex suggesting a role for dynamic protein turnover developing Spemann's organizer. Select only stereotypical embryos for this protocol to improve reproducibility and result interpretation. Dissected tissue should be transferred into a vial and immediately frozen containing as minimal media buffer as possible to minimize contamination from buffer salts.

This approach has enabled us to study protein dynamics in identified cells and cell lineages in live embryos. The new information that we have obtained on the spatial temporal production of important proteins in developing tissues has enabled us to design targeted experiments to assess their biological function.