TITLE: LCM-NanoPOTS workflow for spatial proteome mapping
AUTHORS: Yumi  Kwon, Ernesto S Nakayasu, WEI-JUN  QIAN, Ying  Zhu

[DESCRIPTION]
Spatial proteomics holds great potential to transform our understanding of the role of cell populations and their spatial organizations in human tissues related to diseases. However, in-depth proteome measurement with high spatial resolution has been challenged by the low sample input and inefficient proteomics workflow. 
In this protocol, we outlined the detailed procedure for high-resolution analysis of human pancreas tissue sections using a spatial proteomic platform by combining laser capture microdissection (LCM), nanoPOTS (nanodroplet processing in one pot for trace samples), and nanoflow liquid chromatography mass spectrometry (LC-MS). This protocol has been applied to human pancreas specimens for HubMAP Tissue Mapping Centers. 

Expected outcome:
The unbiased spatial proteome mapping of >3000 proteins at 50-µm spatial resolution depending on sample types and LC-MS instrumentation.

[STEPS]
SECTION: Laser capture microdissection (LCM)
1. Before cut and collection: Load DMSO as capturing media on the nanoPOTS chip
SECTION: Laser capture microdissection (LCM)
1.1. Set the nanoPOTS robot to be ready (Temperature, humidity).




SECTION: Laser capture microdissection (LCM)
1.2. Align the nanoPOTS chip.
SECTION: Laser capture microdissection (LCM)
1.3. Set 100 µL of DMSO (Dimethyl sulfoxide) in the sample plate.
SECTION: Laser capture microdissection (LCM)
1.4. Dispense 200 nL of DMSO into each well on the nanoPOTS chip
SECTION: Laser capture microdissection (LCM)
1.5. Cover the chip with the glass slide and cover with aluminum foil.
SECTION: Laser capture microdissection (LCM)
2. Cut and tissue collection
SECTION: Laser capture microdissection (LCM)
2.1. Turn on the LCM (PALM MicroBeam).
SECTION: Laser capture microdissection (LCM)
2.2. Scan the tissue slide.
SECTION: Laser capture microdissection (LCM)
2.3. Find the target region on scanned image
SECTION: Laser capture microdissection (LCM)
2.4. Navigate to the region of interest (ROI).
SECTION: Laser capture microdissection (LCM)
2.5. Draw 4 × 9 grid lines (1 voxel size= 2500 µm2) on ROI.
SECTION: Laser capture microdissection (LCM)
2.6. Load the laser setting (Speed 1, Cut energy 40, LPC Energy delta15).
SECTION: Laser capture microdissection (LCM)
2.7. Load the nanoPOTS chip on the collector (slide collector 48). Align A1 corner of slide collector and A2 well corner of the nanoPOTS chip.
SECTION: Laser capture microdissection (LCM)
2.8. Start the collection with CenterRoboLPC function.
SECTION: Laser capture microdissection (LCM)
2.9. After collecting all voxels, check on the microscope if every DMSO droplet contains the tissue voxel.
SECTION: Laser capture microdissection (LCM)
2.10. Remove the nanoPOTS chip carefully from thecollector and cover it with the glass slide.
SECTION: Laser capture microdissection (LCM)
3. After collection: Dry and scan
SECTION: Laser capture microdissection (LCM)
3.1. Set incubator temperature to 70 °C.
SECTION: Laser capture microdissection (LCM)
3.5. Wrap with foil and store it in -20 °C until analysis.
SECTION: Laser capture microdissection (LCM)
3.4. After DMSO has evaporated, scan the nanoPOTS chip and check if there is any missing well.
SECTION: Laser capture microdissection (LCM)
3.3. After 15 min, unwrap the chip and place uncovered in incubator for 5 min intervals until DMSO has evaporate (~19 min).
SECTION: Laser capture microdissection (LCM)
3.2. Place chips upside down (still wrapped) into incubator for 15 min.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
4. Extraction
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
5. Alkylation
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
7. Acid Quenching
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
6. Digestion
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
7.1. Remove chips from incubator and place into nanoPOTS robot.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
7.2. Dispense 50 nL of 5% formic acid in water into each well.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
4.1. Set the nanoPOTS robot to be ready (Temperature, humidity).
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
7.4. Cover with a sterile glass slide, wrap with foil and store at -20 °C until analysis.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
4.6. Place the chip upside down in the humidity box in a zipper bag at 70 °C for 60 min.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
4.5. Cover chip with the glass slide and cover with aluminum foil.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
4.4. Dispense 200 nL of the extraction buffer into each well on the nanoPOTS chip.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
4.3. Place 100 µL of extraction buffer (1 mM TCEP (tris(2-carboxyethyl)phosphine), 0.1% DDM (n-Dodecy-ß-D-maltoside), 0.1 M HEPES (pH 8.5)) in the sample plate.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
4.2. Align the nanoPOTS chip.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
5.1. Set chip at RT and put it into nanoPOTS robot.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
5.5. Place the chip upside down in the dark for 30 min at RT.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
5.4. Cover chip with glass slide and wrap with foil.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
5.3. Dispense 50 nL of alkylation buffer (10 mM CAA (2-chloroacetamide) in 0.1 M HEPES) into each well.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
5.2. Place 100 µL of alkylation buffer in the sample plate.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
6.1. Set chip into nanoPOTS robot.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
6.5. Place the chip upside down in the humidity box for 10 h at 37 °C.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
6.4. Cover chip with a glass slide and cover with foil.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
6.3. Dispense 50 nL of digestion buffer (0.01 ng/nL LysC, 0.04 ng/nL trypsin in 0.1 M HEPES) into each well.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
6.2. Place 100 µL of digestion buffer in the sample plate.
SECTION: NanoPOTS Proteomic Sample Processing for LCM tissue
7.3. Remove the chip from the nanoPOTS robot and place it into the desiccator until the droplets have evaporated.
SECTION: NanoPOTS-LC-MS/MS for spatial proteome mapping
8. In-house assembled nanoPOTS autosampler with an in-house packed SPE column (100 μm i.d., 4 cm, 5 μm, 300 Å C18 material, Phenomenex) and an LC column (50 μm i.d., 25 cm, 1.7 μm, 190 Å C18 material, Waters) heated to 50 using AgileSleeve column heater (Analytical Sales and services, Inc., Flanders, NJ) was used for sample analysis.
SECTION: NanoPOTS-LC-MS/MS for spatial proteome mapping
9. Samples were dissolved with Buffer A (0.1% formic acid in water) on the chip, then trapped on the SPE column for 5 min. After washing the peptides, samples were eluted at 100 nL/min and separated using a 60-min gradient from 8% to 35% Buffer B (0.1% formic acid in acetonitrile).
SECTION: NanoPOTS-LC-MS/MS for spatial proteome mapping
10. An Orbitrap Eclipse Tribrid mass spectrometer (Thermo scientific) with FAIMS-pro interface operated in data-dependent acquisition mode was used for all analyses. Peptides were ionized by applying a voltage of 2,400 V.
SECTION: NanoPOTS-LC-MS/MS for spatial proteome mapping
11. The ionized peptides were fractionated by the FAIMSpro interface using a 3-CV (-45, -60, -75 V) method. Fractionated ions with a mass range 350-1600 m/z were scanned at 120,000 resolution with an IT of 118 ms and an AGC target of 1E6.
SECTION: NanoPOTS-LC-MS/MS for spatial proteome mapping
12. For the pooled tissue samples for generating a spectral library, a single CV was used for each LC-MS run. Precursor ions with intensities > 1E4 were selected for fragmentation by 30% HCD and scanned in an Ion trap with an AGC of 2E5 and an IT of 86 ms.
SECTION: Data analysis
13. Use FragPipe (v 17.1, MSFragger v3.4, Philosopher v 4.1.0) for processing (feature detection, database searching and protein/peptide quantification) of all raw files.
SECTION: Data analysis
14. For database search, use the latest Uniprot human database.
SECTION: Data analysis
15. Fixed modification: carbamidomethylation of cysteine / Variable modification: Protein N-terminal acetylation, oxidation of methionine.
SECTION: Data analysis
16. Cleavage enzyme: strict trypsin, peptide length: 7-50, max missed cleavage: 2, FDR 0.01
SECTION: Data analysis
17. For quantifications, use match between runs (MBR) and MaxLFQ embedded in the FragPipe (minimum ions:1; minimum scans: 3; m/z tolerance 10 ppm; RT tolerance 0.4 min; MBR FDR at ion level 0.05).