TITLE: Laser Capture Microdissection of Tissue Functional Units for microPOTS Top-Down Proteomics
AUTHORS: James M Fulcher, Yen-Chen Liao

[DESCRIPTION]
In this protocol, we describe a detailed procedure for high-resolution top-down proteomic analysis of human pancreatic and kidney tissue sections using a spatial proteomic platform by combining laser capture microdissection (LCM), microPOTS (microdroplet processing in one pot for trace samples), and nanoflow liquid chromatography mass spectrometry (LC-MS). This protocol has been applied to human pancreatic and kidney specimens for HubMAP Tissue Mapping Centers. 


Expected outcome:
Identification of >300 proteoforms at 100-µm spatial resolution depending on sample types and LC-MS instrumentation.

[STEPS]
SECTION: Tissue collection
1. 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). 
 

SECTION: Tissue collection
1.1. Pipette 1 µL of DMSO onto each microPOT chip well
SECTION: Tissue collection
2. Tissue section with LCM
SECTION: Tissue collection
1.2. Cover the chip with the glass slide and enclose with aluminum foil. Store at 4°C until ready for use.
SECTION: Tissue collection
2.1. Turn on the LCM (PALM MicroBeam).
SECTION: Tissue collection
2.2. Scan the tissue slide.
SECTION: Tissue collection
2.3. Find the target region on scanned image
SECTION: Tissue collection
2.4. Navigate to the region of interest (ROI).
SECTION: Tissue collection
2.5. Draw grid lines (1 voxel size= 2500 µm2) on ROI.
SECTION: Tissue collection
2.6. Load the laser setting (Speed 1, Cut energy 40, LPC Energy delta15).
SECTION: Tissue collection
2.7. Load the microPOTS chip on the collector (slide collector 48). Align A1 corner of slide collector and A2 well corner of the microPOTS chip.
SECTION: Tissue collection
2.8. Start the collection with CenterRoboLPC function.
SECTION: Tissue collection
2.9. After collecting all voxels, check on the microscope if every DMSO droplet contains the tissue voxel.

SECTION: Tissue collection
2.10. Remove the microPOTS chip carefully from the collector and cover it with the glass slide.

SECTION: Tissue collection
3. Dry DMSO from microPOTS chip
SECTION: Tissue collection
3.1. Set incubator temperature to  70 °C.
SECTION: Tissue collection
3.2. Place uncovered chips  into incubator for 60  minutes
SECTION: Tissue collection
3.3. After DMSO has evaporated, check the microPOTS chip under optical microscope to perform a QC check for missing tissue pieces. 

SECTION: Protein extraction
4. Tissue protein extraction
SECTION: Protein extraction
4.1. Set incubator to 37 °C and place microPOTS chips on ice into a biological safety cabinet (BSC)
SECTION: Protein extraction
4.2. 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.
SECTION: Protein extraction
4.3. Place microPOTS chip with cover slide into incubator at 37 °C for 30 minutes
SECTION: Protein extraction
4.4. Place the microPOTS chip into vacuum chamber and slowly introduce vacuum to evaporate remaining droplet.
SECTION: Protein extraction
5. Nuclease-based histone extraction
SECTION: Protein extraction
5.1. 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). 
SECTION: Protein extraction
5.2. Incubate microPOTS chip with cover slide at  37 °C for 60 minutes
SECTION: Protein extraction
6. Transferring sample for LC-MS analysis
SECTION: Protein extraction
6.1. To an LC-MS vial, add a PCR tube with cap cut off. 
SECTION: Protein extraction
6.3. Transfer 1  µL of sample from each well into a PCR-insert LC-MS vial containing 10 µL of quenching buffer. 
SECTION: Protein extraction
6.2. Add 10  µL of quenching buffer (94.9% water, 4.9% MeCN, 0.2% FA) to each PCR-insert LC-MS vial.
SECTION: Protein extraction
6.4. 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
SECTION: Protein extraction
7. Samples are now prepared and ready for LC-MS analysis.