Sample Preparation for Single Cell Mass Spectrometry Metabolomics Studies: Combined Cell Washing, Quenching, Drying, and Storage

We have developed protocols to preserve the integrity of cellular metabolites for single cell mass spectrometry studies. We investigated how washing, quenching and storage conditions affect cell metabolites. New sample preparation methods and mass spectrometry techniques have been recently developed to advance single-cell metabolomics studies to better understand cellular heterogeneity and disease mechanisms.

Researchers need to overcome multiple key challenges, including limited sample amount, reduced detection sensitivity due to sample complexity and altered metabolites due sample preparation and analysis. We demonstrate comprehensive sample preparation techniques that can preserve cellular metabolites and cell integrity for single cell mass spectrometry metabolomic studies. Our protocols could offer a simple and effective way for preparation, storage and transportation of samples for single cell metabolomic studies using different mass spectrometric techniques.

To begin, incubate the cells at 37 degrees Celsius in a humidified incubator containing 5%carbon dioxide. Monitor the confluency of the cells daily and passage them when cultures reach 80%confluency. For passaging, aspirate the medium from the dish and rinse once with five milliliters of PBS.

Incubate the cells with two milliliters of 0.25%trypsin EDTA at 37 degrees Celsius for three minutes to facilitate cell detachment. Neutralize the trypsin by adding eight milliliters of prewarm complete medium and gently pipette to disperse the cells. Transfer one milliliter of the suspension into nine milliliters of fresh, complete medium for counting the cells.

To seed the cells, dilute them to a final concentration of one times 10 to the power of six cells per milliliter. Place individual 18 millimeter glass cover slips into the wells of a 12 Well plate. Add two milliliters of complete medium and 200 microliters of the cell suspension into each well to achieve two times 10 to the power of five cells per well.

Incubate the cells overnight to allow attachment. For washing the cells, add two milliliters of cold ammonium formate solution to each well of the 12 well plate. Then using sterile forceps, gently lift each cover slip containing adherence cells from its well and briefly place it for two to three seconds in a separate well prefilled with two milliliters of cold 0.9%ammonium formate solution.

Place each ammonium formate rinsed cover slip with the cells facing upward into a petri dish inside an aluminum foil container. Slowly pour 10 to 20 milliliters of liquid nitrogen over the cover slips to ensure complete coverage. And immediately tilt the petri dish with tweezers to decant any remaining liquid nitrogen.

Using cryogenic gloves and insulated tweezers, place the petri dish containing the liquid nitrogen chill cover slips into the vacuum concentrator chamber. Process the samples using the standard vacuum settings for five to seven minutes until the visible liquid nitrogen evaporates and the cover slips appear dry. Ensure that the dried cover slips do not contain any residual liquid nitrogen or ice crystals.

Next, wrap the container with a paper towel and transfer it to a 80 degree Celsius freezer for 48 hours. After storage, transfer the cover slips containing the petri dish into a room temperature desiccate for 10 minutes. Ensure condensed frost or water on the cover slips completely disappears before removing them from the desiccator.

Divide the samples into two groups and treat them appropriately. Now, mount the single probe onto the XYZ stage and affix it to a motorized XYZ manipulator positioned beneath a digital microscope. Couple the single probe setup to a mass spectrometer for SCMS analysis.

Use acetone nitrile containing 0.1%formic acid at a purity of at least 99.9%as the extraction solvent and deliver it at a flow rate of 150 nanoliters per minute using a syringe pump. Set the mass spectrometry parameters for positive and negative ion modes. Now, place the two cover slips from group one and group two together on the motorized XYZ stage of the single probe SCMS setup and use the microscope to randomly select cells for analysis.

After acquiring mass spectra, analyze approximately 30 cells per group using the same single probe, solvent flow rate and ionization voltage. Pre-processed the raw SCMS data using a custom R script and apply background and noise removal followed by ion intensity normalization to total ion current. De-isotope the SCMS data using the Python package MSD isotope.

Perform peak alignment Using an in-house Python script. Apply a bin width of 0.01 master charge ratio for binning and a mass tolerance of 0.01 master charge ratio or five parts per million for peak alignment. Conduct statistical data analysis using Metabo Analyst 6.0.

Perform a T-test and generate a heat map showing relative metabolite levels. Select the option used top 100 and T-test or Inova to generate the top 100 metabolites ranked by significance and plot a heat map using these top 100 metabolites with significant differences. Finally, search the accurate master charge values against the human metabolome database using a mass tolerance of 10 parts per million for database searching.

Principle component analysis in the positive ion mode showed that group one and group two overlapped closely, indicating highly similar metabolite profiles. In the negative ion mode, a comparable pattern was observed, although group two appeared slightly more distinct. Heat map analysis of the top 100 metabolites displayed highly consistent abundance patterns between group one and group two in both ion modes.

No major shifts or group specific clustering are observed. Although minor variations in a few metabolite clusters may reflect differences in ionization efficiency or metabolite stability.