Automated Sample Preparation for the Multiplexed Analysis of Single-Cell Histone Post-Translational Modifications (sc-hPTM2)

We're advancing single cell proteomics with an automated multiplex method to study global chromatin heterogeneity for post-translational modifications. What makes this protocol challenging is the picogram scale proteomic preparation, multiplex labeling, as well as resolving complex combinatorial modified histone peptides. To begin, reconstitute Hep G2/C3A hepatocellular carcinoma cells at a final concentration of 200 to 500 cells per microliter in ice cold PBS.

Set each nozzles pulse width and drive voltage to the values provided by the manufacturer. For maximal pickup efficiency, ensure that the Z offset between the two nozzles differs by less than 50 micrometers. Identify the optimal contact position for each nozzle and compare the Z height readouts in the nozzle setup tab.

For tuplex multiplexing scheme, load a single slide onto the slide holder in the hood and then transfer it to the instrument. Using a pipette, aliquot 150 microliters of LCMS grade DMSO into a new PCR tube. Place it into position two of the wash station with the tube cap facing the instrument door.

Start the DMSO run. Obtain a stable drop once after DMSO has been aspirated. Note the change in the PDC transparency.

It may be required to increase the voltage. Verify stability by performing a drop volume check. Perform the head camera wizard setup to align the camera.

Dispense DMSO and allow the instrument to automatically flush the nozzles and capture images across all slides for quality control. Review these images to verify a uniform DMSO droplet on every slide and to check for missing or off-target droplets. After aspirating the cell suspension, open the cell in one module, perform a background capture and then run the mapping routine to define the ejection zone.

Run the analysis and then gate the particles to select for cells based on size and elongation. Now, prepare a fresh digestion master mix in LCMS grade water. Degas the solution with a vacuum pump for 10 minutes on ice.

After dispensing the digestion mix onto the slides, let the instrument automatically flush the nozzles and photograph every slide. Review the images to verify that each droplet receive the digestion mix uniformly. Incubate the slides at room temperature overnight to perform digestion.

After starting the evaporation run and overnight digestion, inspect the images. If the droplets are sufficiently dry, stop the run. If not, click continue and dry for another five minutes.

For labeling, first prepare stock solutions for multiplexed histone derivitization. After dispensing, allow the instrument to automatically flush the nozzles and photograph every slide. Review the images to confirm even distribution and correct droplet placement across all slides.

After incubation is complete, dispense hydroxylamine solution for quenching. To pick up the sample, select the run in the main tab, and then start the run in the run tab. After the pickup is finished, review the images to check for any errors.

Once done with pickup, remove the cover from the pickup plate. Then dry the samples at low heat in a vacuum concentrator. For data acquisition, program the high performance liquid chromatography method.

Set the MS two experiment to contain isolation windows of 50 mass overcharge, high energy collision dissociation of 27%and automatic gain control target of 1000%Cover the plate with a rubber seal mat and place it inside the auto sampler. After obtaining raw files, briefly check the chromatogram and then run the raw data in EpiProfile version 2.1. Review the output table of normalized histone PTM abundances and use it for downstream analyses.

The first 20 histone H3 peptidoforms were quantified, displaying their relative abundances in control and sodium butyrate treated conditions. Both H3 K9 acetylation, and H3 K 14 acetylation displayed higher relative abundance upon sodium butyrate treatment. While H3 K9 acetylation K14 acetylation appeared even more enriched in treated cells.

Total acetylation levels increased following sodium butyrate treatment with treated cells showing a broader error distribution compared to controls reflecting the higher heterogeneity captured at the single cell level. The quantification of single and co-occurring histone modifications demonstrated that combinatorial acetylation states were most strongly affected by sodium butyrate. The wider dispersion of sodium butyrate treated cells indicated increased biological heterogeneity among individual cells within the spheroids.

This protocol closes the gap in epigenetic research by allowing scientists to study chromatin states at single cell solution. This workflow enables high throughput sample preparation, sample multiplexing, and unbiased mass spectrometry to produce both sensitive and quantitative single cell epigenetic data. Future research will explore how the regulation of the chromatin is altering disease states such as cancer, metabolic disease, and aging.