Platform Incubator with Movable XY Stage: A New Platform for Implementing In-Cell Fast Photochemical Oxidation of Proteins

1. Assembly of Platform Incubator with Movable XY stage

NOTE: The new platform includes the incubation system, the positioning stage and controllers, the peristaltic pumps, the 248 nm KrF excimer laser, and the optical mirrors assembled on an Imperial optical breadboard.

1. Assemble the incubation system: the temperature unit, carbon dioxide unit, humidifier, air pump, and touch monitoring system (Figure 2A-E).
   NOTE: Detailed assembly instructions are provided by the manufacturer. The incubation system must stabilize to 5% CO₂, 37 °C, and 85% humidity before growing cells within the incubator. These parameters may depend on the cell line.
2. Assemble the custom six-well plate incubator, nanopositioning drive stage, and XY drive stage. The former screws into the latter, respectively.
   NOTE: Detailed assembly instructions provided by the manufacturer.
3. Connect the four peristaltic pumps in a "daisy chain" sequence via RS-232 cables, with an RS-232 to USB cable connected to the controlling computer. Connect 3.18 mm ID polymer tubing (e.g., Tygon) to every channel on each pump channel roller.
   NOTE: Each pump has four rollers. Direction and flow rate of rollers are manually manipulated.
4. Insert 1/16" x 1/8" connectors at the end of each 3.18 mm ID polymer tube. Insert the 1/16'' end of the connector into 1.59 ID polymer tubing, then insert the polymer tubing into the incubator via custom ports. Place the other end of the tube in the solution that will be infused during IC-FPOP experimentation.
   NOTE: For the perfusion lines, the incubator has 36 custom ports for tubing lines all around the periphery to accommodate all the reagents used.
5. Screw one 50 mm, 248 nm, 45° excimer laser line mirror within a kinematic mirror mount for Ø2" optics into the breadboard 10-11 grid points from the 248 nm laser aperture. Place the second mirror at a 90° angle to the first on the other side of the incubator. Angle the second mirror downward at approximately 45° for laser beam guidance to the incubator (Figure 2F-J).

2. Synchronization and initial automation of system via integration software

1. Install the latest version of the integration software needed to control the drivers and pumps.
2. Referring to the pump manual, rename the pumps starting with '5' and increasing in value. The commands can be sent to the pump system using the Manual Control sub-program in the integration software.
   NOTE: Setting a pump to Channel Mode automatically changes the pump naming convention into the set, 1, 2, 3 and 4, corresponding to the four pump channels.
3. Open the integration software program for the automation of the platform incubator.
4. Choose the appropriate USB comport device name (e.g., COM4) corresponding to the pump system USB cable from the connection dropdown menu labelled Comport for pumps.
5. Click the Script Builder button to edit/create a script for the automated platform incubator platform. Click Save Script to save the sequence as a text file (Figure 3A).
   NOTE: The script builder has a user interface for defining the script with definitions of pump number, direction, rate, volume, tubing diameter, channel mode, and timed delay.
6. If channel mode is desired in the making of the script, a step in the script must be dedicated to changing the pump in and out of channel mode, and the following steps corresponding to the pump channels must be labelled as pumps 1-4.
   NOTE: This ensures that no two pumps can simultaneously be in channel mode during a platform incubator script and that each pump is switched back to legacy mode after the commands have been sent to the needed channels.
7. Click the Read Script button and choose the appropriate script file desired for the platform incubator operation.
8. Switch the Run Sequence button to the ON position (green) to run the script and then click the START button (Figure 3B) .

3. Grow cells in the platform incubator

NOTE: Cells must be placed in the platform incubator under sterile conditions in a cell culture hood the day before experimentation.

1. Unscrew the platform incubator from the nanopositioning stage and disconnect the temperature, gas, and humidifier lines. After spraying the incubator with 70% ethanol, place it in a cell culture hood.
2. Grow cells in a T-175 to about 80-90% confluency prior to transfer.
   NOTE: The term confluency is used as a measure of the number/coverage of the cells in a cell culture dish or a flask.
3. Remove media and rinse with buffer.
4. Detach cells using trypsin-EDTA using manufacturer's protocol.
5. Resuspend in 8 mL of media and count the cells.
6. Seed fibronectin/collagen¹⁴ coated six-well plates with approximately 90-95 μL of resuspended cells in each well. This volume will be appropriate to achieve 80-90% confluency (~1.2 million cells) in each well on the next day.
   NOTE: Six-well plates must be coated with collagen prior to seeding. The reagents used during IC-FPOP are infused with fast flow rates. Coated plates ensure cells are not prematurely detached due to infusion of reagents.
7. Place the seeded plate into the platform incubator and cover with the quartz lid.
   NOTE: During design and development, a glass incubator lid was changed to quartz, so it is compatible with the ultraviolet laser light.
8. Replace and secure the platform incubator back on the nanopositioning drive stage. Reconnect the temperature, gas, and humidifier lines.
9. Let cells grow to confluency overnight.
   NOTE: The following cell culture steps are optional and are intended for experiments in which human cells are transiently transfected. These steps assess transfection efficiency under cell culture conditions.
10. Transfect the plasmid containing the gene for the chimeric protein GCaMP2 into HEK 293 cells using a commercial cationic-lipid transfection kit.
11. Perform transient transfections of GCaMP2 in HEK293T cells in two six-well plates.
12. Incubate one six-well plate in the platform incubator, and the second six-well plate in a standard CO₂ incubator.
13. Compare transfection efficiency within each plate by fluorescence imaging using a fluorescence microscope.

4. Make quench buffer and H₂O₂

1. Make 100 mL of quench buffer containing 125 mM N-tert-Butyl-α-phenylnitrone (PBN) and 125 mM N, N′-Dimethylthiourea (DMTU).
   NOTE: DMTU and PBN are free radical scavengers and are cell permeable.
2. Dilute H₂O₂ to 200 mM. Each sample requires 6 mL of H₂O₂ to fully immerse the layer of cells at the bottom of each well.
   ​NOTE: The quench buffer is made the day before and stored at 4 °C overnight, protected from light. Dilute the H₂O₂ on the day of the experiment.

5. Set up the platform incubator for IC-FPOP

NOTE: The platform incubator must be assembled under sterile conditions. Assemble the incubator in a sterile cell culture hood.

1. Unscrew the platform incubator from the positioning stage and disconnect the temperature, gas, and humidifier lines.
2. After spraying the incubator with 70% ethanol, place it in the cell culture hood.
3. Remove the six-well plate from the platform incubator, secure the plate's original lid, and confirm cell confluency using a microscope.
4. After cell confluency has been confirmed, replace the six-well plate with confluent cells back in the platform incubator inside the cell culture hood.
5. Insert three precut (15 cm) 1.59 ID polymer tubes in each well via the embedded ports. Flush the tubes to the well walls and hold with custom 3D printed rings.
   NOTE: Six 33 mm PLA filament 3D printed rings were custom designed to secure the tubing to the walls of the plate without disturbing the cells or getting in the way of the laser pulse.
6. Cover the platform incubator with its quartz lid. Replace and secure the stage top incubator on the positioning system. Reconnect the temperature, gas, and humidifier lines.
7. Connect 1.59 ID polymer tubing to 1/16" end of the 1/16×1/8" connectors.

6. Performing IC-FPOP in the platform incubator

1. Prepare an integration software script for pump withdrawal. Use one peristaltic pump (Pump 8) to completely remove cell media from all six wells.
2. Prime solvents in each channel of the other three peristaltic pumps (Pumps 5-7). Infuse H₂O₂ and quench buffer in their respective alternating tubes until the reagents reach the incubator ports.
3. Confirm that the laser beam has been angled correctly by the mirrors and reaches the incubator uninhibited.
   NOTE: Laser safety goggles must be worn whenever the laser is in use. Do not prematurely irradiate the cells during the angling/alignment process. Use cardboard to completely cover the quartz lid when aligning the beam. Also, use a printed outline on white paper of a six-well plate to further confirm the laser beam is hitting the center of each well. Use the Continuous pulse setting at the lowest frequency and energy for alignment.
4. Check laser energy using an external sensor. Use one single laser pulse of 160 mJ at 50 Hz and 27 kV.
   NOTE: A timer is needed for the following steps.
5. Prepare the integration software script for pump infusion after confirming beam alignment.
6. Begin the timer and press the Start button on the integration software pump script at the same time.
7. Infuse 200 mM H₂O₂ at 35 mL/min into the first well (6-10 second mark on timer).
   NOTE: There is a five second delay before a pump begins to infuse. It also takes the laser seven seconds before the pulse is triggered. The laser pulse must come immediately after H₂O₂ infusion.
8. Press the Start button on the laser software at the 5 second mark to trigger the pulse at the 11 second mark on the timer.
9. Infuse 125 mM quench solution at 35 mL/min into the first well immediately after the laser pulse. Manually move the positioning stage to align the next well with the laser beam.
10. Repeat the above steps 6.5-6.9 until each well has been processed.
    NOTE: IC-POP is performed in technical triplicate of three laser and three non-laser samples. One processed six-well plate serves as one biological replicate.
11. In a cell culture hood, use a cell scraper to transfer the cells from each well into individual 15 mL conical tubes. Centrifuge cells at 1,200 x g for 5 minutes.
12. Discard the supernatant and resuspend cells in 100 μL of RIPA lysis buffer.
13. Transfer cells to individual 1.2 mL polypropylene tubes.
14. Flash freeze all samples in liquid nitrogen and place in a -80 °C freezer until use.
    ​NOTE: The protocol can be paused here.

7. Protein extraction, purification, and proteolysis

1. Thaw the samples and heat at 95 °C in a heat block for 5 minutes.
2. After heating, cool on ice for 5 minutes.
3. Add 25 units of nuclease to the cell lysate to degrade single-stranded, double-stranded, linear and circular DNA and RNA and incubate at room temperate for 15 minutes.
4. Centrifuge samples at 16,000 x g for 10 minutes at 4 °C.
5. Collect the supernatant and transfer to a clean polypropylene tube.
6. Check the protein concentration using a colorimetric protein assay.
7. Transfer 100 µg of sample to a clean polypropylene tube and bring to 100 µL with cell lysis buffer.
8. Reduce samples with 10 mM dithiothreitol (DTT) at 50 °C for 45 minutes.
9. Cool samples at room temperature for 10 minutes.
10. Alkylate with 50 mM iodoacetamide (IAA) at room temperature for 20 minutes.
    NOTE: Protect IAA from light
11. Add 460 µL of pre-chilled (-20 °C) acetone. Vortex samples and place at -20 °C overnight.
    NOTE: The protocol can be paused here.
12. Next day, centrifuge samples at 16,000 x g for 10 minutes at 4 °C.
13. Remove acetone without disrupting the protein pellet.
14. Add 50 µL of 90% pre-chilled (-20 °C) acetone. Vortex samples to mix and centrifuge at 16,000 x g for an additional 5 minutes at 4 °C.
15. Remove acetone and let samples dry for 2-3 minutes.
16. Resuspend protein pellet with 10 mM Tris buffer pH 8.
17. Resuspend MS grade trypsin (20 µg stock) in 40 µL of 10 mM Tris buffer pH 8 and add 2.5 µg of trypsin to each sample.
18. Incubate samples at 37 °C overnight.
    ​NOTE: The protocol can be paused here.
19. Assess peptide concentration using a colorimetric peptide assay.
20. Quench the samples with 5% formic acid.
21. Transfer 10 µg of sample to a clean polypropylene tube.
22. Dry the sample using a vacuum centrifuge and resuspend with 20 µL of MS grade 0.1% formic acid (FA) in water.
23. Transfer each sample to clean autosampler vials with pre-slit caps.

8. High performance Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

1. To localize FPOP modifications, analyze the digested cell lysate using LC-MS/MS analysis.
2. Use mobile phases of 0.1% FA in water (A) and 0.1% FA in acetonitrile (ACN) (B).
3. Load 0.5 µg of sample onto a 180 µm x 20 mm C18 (5 µm and 100 Å) trapping column and wash the column with 99% (A) and 1% (B) for 15 minutes.
4. Using a 75 µm x 30 cm C18 (5 µm and 125 Å) analytical column, elute and separate digested peptides with a flow rate of 0.300 µL/min for 120 minutes.
5. Run LC gradient as follows: 0−1 min, 3% solvent B; 2−100 min, 10−45% B;100−110 min, 45-100% B; 110−115 min, 100% B.
6. Recondition the column at 3% (B) from 115-116 minutes and hold at 3% (B) from 116-120 minutes.
7. Analyze eluted peptides in positive ion mode with nano electrospray ionization.
8. Acquire MS1 spectra over a m/z scan range of 375-1500 at a resolution of 60,000.
9. Set the automatic gain control (AGC) target to 5.0e⁵ with a maximum injection time of 50 ms and 5.0e⁴ intensity threshold.
10. Isolate precursor ions with charge states 2-6 via data dependent acquisition (DDA) with an isolation window of 1.2 m/z and a cycle time of 4 seconds.
11. Subject MS2 ions to high-energy collisional dissociation (HCD) (32% normalized energy).
12. Exclude peptides after 1 MS/MS acquisition for 60 seconds.
13. Set MS/MS resolution to 15,000 with an AGC target of 5.0e⁴ and a maximum injection time of 35 ms.

9. Proteome discoverer/data processing

1. Search tandem raw data files on available bottom-up proteomics analysis software against a relevant Homo sapiens protein database and digest enzyme.
2. Set the protein analysis search parameters.
3. Set fragment tolerance to 0.02 Da and parent ion tolerance to 10 ppm.
4. Set enzyme specificity to trypsin and allow for one missed cleavage.
5. Set mass range to 375-1500 m/z.
6. Establish peptide confidence at 95% (medium) and residue confidence at 99% (high).
7. Accept proteins if at least two distinct peptides are identified with the 5% discovery rate (FDR) filter.
8. Set carbamidomethylation as a static modification and all known hydroxyl radical side-chain modifications^15,16 as dynamic modifications.
9. Once files are finished searching, export sequence, modification locations, protein accession, spectrum file, precursor abundance, and retention time information to an electronic database.
10. Calculate the extent of modification per peptide or residue from this equation:
    
    NOTE: EIC area modified is the extracted ion chromatographic area (EIC) of the peptide or residue with an oxidative modification, and EIC area is the total area of the same peptide or residue with and without the oxidative modification. Over time, protein in the presence of hydrogen peroxide will oxidize, resulting in background oxidations. To calculate the extent of modification, the area of the modified peptide is divided by the total area. A non-irradiated control sample accounts for background oxidation. The background oxidation from the control sample is subtracted out from the laser treated sample to identify an FPOP modification.