Aug 12, 2022
Laser Capture Microdissection of Tissue Functional Units for microPOTS Top-Down Proteomics
- 1Pacific Northwest National lab;
- 2Pacific Northwest National Laboratory
Protocol Citation: James M Fulcher, Dusan Velickovic, Yen-Chen Liao, Ying Zhu, Mowei Zhou, Ljiljana.PasaTolic 2022. Laser Capture Microdissection of Tissue Functional Units for microPOTS Top-Down Proteomics. protocols.io https://dx.doi.org/10.17504/protocols.io.ceu5tey6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 04, 2022
Last Modified: August 12, 2022
Protocol Integer ID: 68221
Keywords: laser capture microdissection, tissue
Funders Acknowledgement:
National Institutes of Health (NIH) Common Fund, Human Biomolecular Atlas Program (HuBMAP)
Grant ID: UG3CA256959-01
Abstract
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.
Materials
1. Reagents
1.1. LCM collection
• Dimethyl sulfoxide (DMSO)
1.2. Proteomic sample processing
• Nanopure water
• LC-MS grade DMSO
• n-Dodecy-ß-D-maltoside (DDM) (e.g. Thermo Fisher Scientific Cat# 89902)
• Ammonium bicarbonate (ABC)
• Magnesium chloride (MgCl2)
• 250 units/µL benzonase (EMDMillipore, Cat# 71206-3)
• 0.5 M Neutral pH, TCEP (tris(2-carboxyethyl)phosphine)-HCL (Thermo Fisher scientific, Cat# 77720)
• Urea BioUltra (Sigma Aldrich, SKU 51456-500G)
• Formic acid (Thermo Fisher scientific, Cat# 28905)
• Acetonitrile (MeCN)
2. Equipment
• Laser Capture Microdissection System (Zeiss PALM MicroBeam)
• Home-built nanoPOTS sample preparation system
• polypropylene microPOTS chips
CITATION
• LC-MS/MS system (Orbitrap Lumos Tribrid MS, Thermo Scientific)
3. Buffer preparation
3.1. Extraction buffer (Desired conc.: ~4 M Urea, 10%DMSO, 5 mM TCEP, 0.1% DDM in 50 mM ABC)
• Weight 240 mg urea into 1.5 mL Eppendorf Tube
• Add 530 µL 50 mM ABC (pH 7.8)
• 100 µL 100% LC-MS grade DMSO
• 100 µL 1% DDM
• 10 µL 0.5 M TCEP
3.2. Benzonase buffer (Desired conc.: 1 mM MgCl2 in 50 mM ABC with 2.5 unit/µL benzonase)
• 98 µL of 50 mM ABC
• 1 µL of 100 mM MgCl2
• 1 µL of 250 units/µL benzonase
3.3. Quenching buffer (Desired conc.: 94.9% water, 4.9% MeCN, 0.2% FA)
• 949 µL LC-MS grade water
• 49 µL MeCN
• 2 µL formic acid
Tissue collection
Tissue collection
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).
CITATION
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).
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.
Remove the microPOTS chip carefully from the collector and cover it with the glass slide.
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.
Protein extraction
Protein extraction
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.
Citations
Weke K, Singh A, Uwugiaren N, Alfaro JA, Wang T, Hupp TR, O'Neill JR, Vojtesek B, Goodlett DR, Williams SM, Zhou M, Kelly RT, Zhu Y, Dapic I. MicroPOTS Analysis of Barrett's Esophageal Cell Line Models Identifies Proteomic Changes after Physiologic and Radiation Stress.
https://doi.org/10.1021/acs.jproteome.0c00629Step 1
Xu K, Liang Y, Piehowski PD, Dou M, Schwarz KC, Zhao R, Sontag RL, Moore RJ, Zhu Y, Kelly RT. Benchtop-compatible sample processing workflow for proteome profiling of < 100 mammalian cells.
https://doi.org/10.1007/s00216-018-1493-9