Laser Capture Microdissection of Tissue Functional Units for microPOTS Top-Down Proteomics

Load DMSO as capturing media onto the microPOTS chip. Microdroplet processing in one pot for trace samples (microPOTS) chips are polyproplyene chips designed for benchtop manipulation of low sample volumes ( < 5 μL).

Pipette 1 µL of DMSO onto each microPOT chip well

Cover the chip with the glass slide and enclose with aluminum foil. Store at 4°C until ready for use.

Tissue section with LCM

Turn on the LCM (PALM MicroBeam).

Remove the microPOTS chip carefully from the collector and cover it with the glass slide.

Scan the tissue slide.

Find the target region on scanned image

Navigate to the region of interest (ROI).

Draw grid lines (1 voxel size= 2500 µm2) on ROI.

Load the laser setting (Speed 1, Cut energy 40, LPC Energy delta15).

Load the microPOTS chip on the collector (slide collector 48). Align A1 corner of slide collector and A2 well corner of the microPOTS chip.

Start the collection with CenterRoboLPC function.

After collecting all voxels, check on the microscope if every DMSO droplet contains the tissue voxel.

Dry DMSO from microPOTS chip

Set incubator temperature to 70 °C.

Place uncovered chips into incubator for 60 minutes

After DMSO has evaporated, check the microPOTS chip under optical microscope to perform a QC check for missing tissue pieces.

Tissue protein extraction

Set incubator to 37 °C and place microPOTS chips on ice into a biological safety cabinet (BSC)

Add 1 µL of extraction buffer (4 M Urea, 5 mM TCEP, 10% DMSO, 0.1% DDM in 50 mM ABC) to each well containing tissue.

Place microPOTS chip with cover slide into incubator at 37 °C for 30 minutes

Place the microPOTS chip into vacuum chamber and slowly introduce vacuum to evaporate remaining droplet.

Nuclease-based histone extraction

To each well on the microPOTS chip, add 1 µL of benzonase buffer (1 mM MgCl₂ in 50 mM ABC with 2.5 unit/µL benzonase).

Incubate microPOTS chip with cover slide at 37 °C for 60 minutes

Transferring sample for LC-MS analysis

To an LC-MS vial, add a PCR tube with cap cut off.

Add 10 µL of quenching buffer (94.9% water, 4.9% MeCN, 0.2% FA) to each PCR-insert LC-MS vial.

Transfer 1 µL of sample from each well into a PCR-insert LC-MS vial containing 10 µL of quenching buffer.

Re-pipette 2 µL of quenching buffer from the LC-MS vial back into the well to extract protein that may be adsorbed to the microPOTS surface. Remove the 2 µL back into the PCR-insert LC-MS vial

Samples are now prepared and ready for LC-MS analysis.