Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

1. Set up IC-FPOP flow system

1. To begin the assembly of the flow system, cut the fused silica using a cleavage stone to size. The IC-FPOP flow system requires four 12 cm and one 17 cm fused silica with an inner diameter (ID) of 450 µm and outer diameter (OD) of 670 µm, two 24 cm and one 40 cm with an ID of 75 µm and an OD of 360 µm, and finally two 57 cm with an ID of 150 µm and an OD of 360 µm.
   NOTE: When cutting the silica tubing, gently scrape away the polyimide coating, and bend to get the cleanest cut. Check to make sure it is a straight cut (this is necessary to ensure no blockages or leaks form).
2. Set up 15 connections using nano-tight sleeves (0.0155" ID X 1/16" OD for 360 µm OD silica tubing and 0.027" ID X 1/16" OD for the 670 µm OD silica tubing) with super flangeless nut PEEK 1/4-28 flat-bottom for 1/16" OD and super flangeless ferrule w/SST ring, Tefzel (ETFE), 1/4-28 flat-bottom, for 1/16" OD. Construct connections as per the manufacturer's protocol (Figure 1).
3. Place 6 small cylindrical magnets in one 500 µL syringe. Fill this syringe along with another 500 µL syringe and two 5 mL syringes with buffer and remove air. Position on syringe pump as shown on Figure 2A.
   NOTE: The 5 mL syringes are larger than the 500 µL syringes, so a spacer is needed to tighten all the syringes simultaneously in place (Figure 2B).
4. Tighten syringe pump stopper so that the cell syringe has roughly 50 µL left when the motor stalls. This will leave room for the magnetic stirrers. (Figure 2C-D).
   CAUTION: If the syringe pump puts pressure on the magnets, they will jam the syringe and can cause the syringe to break.
5. Using a Luer adapter, connect a manual valve to each syringe. Assemble the silica tubing as shown in Figure 3.
   NOTE: Thread the line with the cells + H₂O₂ all the way through the cross to the other side. Then insert it into the 450 µm ID silica tubing. The sheath buffers in the 5 mL syringe will be flowing at a faster rate than the cells and H₂O₂. With the sheath buffers on both sides, the cells will be hydrodynamically focused into a single line for irradiation.
6. Position flow system next to laser. Using a lighter, burn away the silica coating on the 450 µm ID tubing to make a window for laser irradiation.
7. Place a magnetic stirrer above the cell syringe containing the six magnets.
8. Set syringe pump to 492.4 µL/min for a final flow rate of 1,083.3 µL/min. Flow buffer through the system three times to flush the system and test for any leaks.
9. Focus the excimer laser onto the silica tubing using a convex lens. Once focused, test the irradiation window by placing a small piece of paper behind the silica tubing and turn the laser on. Measure the region burnt from the irradiation. Calculate the needed laser frequency by using the irradiation window and flow rate to obtain an exclusion fraction of zero.
   NOTE: The recommended laser energy for an IC-FPOP experiment is ≥120 mJ. To get an exclusion fraction of 0 with an irradiation window of 2.58 mm and a flow rate of 1083.3 µL/min, the frequency needs to be 44. Below are the equations that are needed to calculate the laser frequency if the irradiation window, flow rate, and exclusion fraction is known.
   
   where w is the volume in nL, x is the laser spot width in mm, y is the silica tubing ID in µm, v is the total volume in µL, b is the exclusion fraction, a is the flow rate in µL/min, and f is the frequency in Hz.

2. Make quench and H₂O₂

1. Make quench containing 100 mM N-tert-Butyl-alpha-phenylnitrone (PBN) and 100 mM N,N'-dimethylthiourea (DMTU). Aliquot 11 mL of quench for each sample into a 50 mL conical tube.
2. Dilute H₂O₂ to 200 mM. Each sample requires 500 µL of H₂O₂.
   NOTE: The quench can be made the day before and stored at 4 °C overnight, protected from light. H₂O₂ should be made fresh the day of experimentation.

3. Collect cells

1. Grow cells in a T175 flask to about 70-90% confluency.
2. Remove media and rinse with buffer.
   NOTE: Typical buffers to use are cell culture grade Dulbecco's phosphate-buffered saline (DPBS) or Hank's balanced salt solution (HBSS).
3. Detach cells using either trypsin-EDTA or with a scraper.
4. Once detached resuspend in 10 mL of buffer and count the cells.
5. Spin down, remove the buffer and trypsin-EDTA, and resuspend to make 2 x 10⁶ cells/mL.
6. Aliquot 500 µL of the cells per sample.
   NOTE: For each condition, make a minimum of 3 laser samples, and 3 no laser controls.

4. Performing IC-FPOP

1. Fill the two 5 mL syringes with buffer, the 500 µL syringe containing the magnets with the cells, and the final 500 µL syringe with H₂O₂. Turn the magnetic stirrer on.
2. Spike in 220 µL of dimethyl sulfoxide (DMSO) to one aliquot of quench, gently mix, and place behind flow system to collect irradiated samples. The addition of DMSO will inhibit endogenous methionine sulfoxide reductase.
3. Turn on laser, wait 7 s, and then turn on flow system.
4. Once the sample finishes flowing, turn the laser off, and gently mix the quench with the collected sample. Place this to the side during steps 4.5 and 4.6.
5. Fill all four syringes with the buffer the cells are suspended in and flow it through the flow system.
6. After the system finishes flushing, repeat steps 4.1 and 4.2. Start the flow without irradiation. This is the no laser control to account for background oxidation in the cells.
7. While the next sample is running, spin down the previous sample at 450-800 x g for 5 min, remove the solvent, and resuspend in 100 µL of a cell lysis buffer like radioimmunoprecipitation assay (RIPA) buffer.
8. Transfer the sample to a microcentrifuge tube and flash freeze in liquid nitrogen.
9. When all samples have finished running, disassemble the flow system for cleaning. Discard the used silica tubing and clean all the other connections by sonicating for 1 h in 50% water: 50% methanol. Clean the syringes as per manufacturer's instructions.

5. Digest

1. Digest the whole cell lysate. Begin by thawing the samples and heat at 95 °C for 10 min.
2. After heating, cool the lysate on ice for 15 min.
3. Add 25 units of nuclease to digest DNA and RNA and incubate at room temperate for 15 min.
4. Spin samples using a table-top centrifuge at 16,000 x g for 10 min at 4 °C.
5. Collect the supernatant and transfer it to a clean microcentrifuge tube.
6. Check the protein concentration using a protein assay kit.
7. Transfer 20-100 µg of sample to a clean microcentrifuge tube and bring to 100 µL.
8. Reduce samples with 20 mM dithiothreiotol (DTT) at 50 °C for 45 min.
9. Cool the samples at room temperature for 15 min.
10. Alkylate with 20 mM iodoacetamide (IAA) at room temperature for 20 min protected from light.
11. Add pre-chilled acetone at a 1:4 ratio protein: acetone. Mix samples and place in -20 °C overnight.
12. The next morning, spin samples at 16,000 x g for 10 min at 4 °C.
13. Remove the supernatant and add 50 µL of 90% pre-chilled acetone. Mix samples and spin down at 16,000 x g for 5 min at 4 °C.
14. Remove acetone and let samples dry by leaving the caps of the microcentrifuge open with a lint free wipe covering the top. After the samples have dried, resuspend protein pellet with 10 mM Tris buffer pH 8.
15. Resuspend 20 µg of trypsin in 40 µL of 10 mM Tris buffer pH 8. Add 2 µg of trypsin (mass: mass ratio of 1 trypsin: 50 sample). Incubate samples at 37 °C overnight.
16. The next morning check the peptide concentration using a peptide assay. After the sample has been removed for the peptide assay, quench the trypsin digestion by adding formic acid to the samples (final concentration is 5% formic acid).
17. Once final peptide concentration is determined, transfer 10 µg of each sample to a clean microcentrifuge tube. This ensures the same amount of each sample is analyzed. Dry the sample using a vacuum centrifuge. Once dried resuspend with 20 µL of mass spectrometry grade 0.1% formic acid. Transfer samples to autosampler vials.

6. Liquid Chromatography-Tandem Mass Spectrometry

1. To localize FPOP modifications analyze the digested cell lysate using LC-MS/MS analysis.
2. Use mobile phases of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (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 sample with 99% (A) and 1% (B) for 15 min.
4. Using a 75 µm x 30 cm C18 (5 µm and 125 Å) analytical column, run the analytical separation method with a flow rate of 0.300 µL/min starting at 3% (B) for one min then ramp to 10% (B) from 1-2 min. Next ramp to 45% (B) from 2-100 min then 100% (B) from 100-110 min. Clean the column by holding at 100% (B) from 110-115 min. Recondition the column by ramping down to 3% (B) from 115-116 min and hold at 3% (B) from 116-130 min.
5. Set the MS acquisition method to have a resolution of 60,000 with an m/z scan range of 375-1500. Set the automatic gain control (AGC) target to 5.0 x 10⁵ with a maximum injection time of 50 ms.
6. During the MS acquisition, select the precursor ions with charge states 2-6 for isolation via data dependent acquisition (DDA) with an isolation window of 1.2 m/z and a cycle time of 4 s. Select the peptides with an intensity threshold of 5.0 x 10⁴ for HCD activation with a normalized energy set to 32%. Exclude the peptides after 1 MS/MS acquisition for 60 s. Set the MS/MS resolution to 15,000 with an AGC target of 5.0 x 10⁴ and a maximum injection time of 35 ms.

7. Data Processing

1. Search the RAW files on an available protein analysis software against a relevant protein database and the relevant digest enzyme. Here, use the Swiss-Prot Homo Sapiens database and trypsin.
2. Set the precursor mass to search between 350 to 5,000 Da and a mass tolerance of 10 ppm. There can be at most 1 missed cleavage site with a peptide length between 6 to 144 residues. These limitations facilitate database searching.
3. Set the fragment ions maximum mass tolerance to 0.02 Da with the carbamidomethyl (+57.021) as a static modification and all FPOP modifications from 17 amino acids as a dynamic modification (Figure 4 is an example of the protein analysis workflow used to detect FPOP modifications).
   NOTE: FPOP modifications on serine and threonine are not included in the search due to their lower reactivity with hydroxyl radicals.
4. Search with a decoy database with a false discovery rate of 1% and 5%.
5. Once files are finished searching calculate the extent of FPOP modification. Open Proteome Discoverer 2.2, export sequence, modification locations, protein accession, spectrum file, precursor abundance, and retention time information. Calculate the extent of modification from equation 4:
   
   The EIC area modified is the chromatographic area of a specific peptide with a hydroxyl radical modification. The EIC area is the total chromatographic area of both modified and unmodified areas of that specific peptide. The extent of oxidation is calculated in the samples with and without irradiation. The sample omitting irradiation (control) accounts for any background oxidation that could have been present in the cells before and after H₂O₂ exposure. The controls are subtracted from the irradiation samples to help isolate FPOP specific modifications.