Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes

1. Prepare the Optical Bench and the Capillary for FPOP

CAUTION: KrF excimer lasers are extreme eye hazards, and direct or reflected light can cause permanent eye damage. Always wear appropriate eye protection, avoid the presence of any reflective objects near the beam path when possible, and use engineering controls to prevent unauthorized access to an active laser and to restrain any stray reflections.

1. Prepare the FPOP optical bench.
   1. Turn on the laser to warm up. Set the laser to External Trigger, Constant Energy, No Gas Replacement. Set the laser energy per pulse (typically between 80-120 mJ/pulse).
   2. Set up the optical bench with the plano-convex lens (30 mm Dia. x 120 mm FL uncoated) directly in the path of the laser beam and a non-reflective backstop to absorb the light as shown in Figure 3A.

Figure 3: Optical bench for the FPOP experiment. (A) The sample is mixed with H₂O₂, adenine radical dosimeter, and glutamine scavenger and loaded into the syringe. The sample is pushed through the fused silica capillary through the focused beam path of a KrF excimer UV laser. The UV light photolyzes H₂O₂ into hydroxyl radicals, which oxidizes the protein and adenine dosimeter. The syringe flow pushes the illuminated sample out of the path of the laser before the next laser pulse, with an unilluminated exclusion volume between illuminated regions. Immediately after oxidation, the sample is passed through an inline UV spectrophotometer, which measures the UV absorbance of adenine at 265 nm. The sample is then deposited into a quench buffer to eliminate the remaining H₂O₂ and secondary oxidants. (B) The spot size is measured after irradiating a colored sticky note affixed behind the capillary with the laser at 248 nm. The width of the spot is used for calculating the sample flow rate, and the silhouette of the capillary in the center of the spot is used to align the optical bench. Please click here to view a larger version of this figure.

2. Cut an appropriate length of the fused silica capillary (360 µm outer diameter and 100 µm inner diameter) and using a sleeve, connect to the gas-tight syringe using a low dead volume connector.
3. Gently burn the polyimide coating of the capillary with a butane torch at the place where the inline dosimeter reads the absorbance signal at 265 nm after laser exposure of the samples. Wipe the debris on the capillary gently using methanol on a lint-free wipe. The polyimide coating at the site of laser incidence can either be similarly burned off with the butane torch or burned off with the excimer laser firing at low power.
   NOTE: Wait for the capillary to cool as it is a fire hazard to use the methanol on the hot capillary.
4. Place this capillary through the beam path of the laser and into the inline dosimeter.
   1. Press the lever on the top of the inline dosimeter to open the hinge. Remove the magnetic holders. Place the capillary in the machined groove of the inline dosimeter, using the magnetic holders to keep the capillary in place. Close the dosimeter hinge over the capillary, pressing it until the lever locks in place.
5. Using the dosimetry software, click on the Start Flash button to begin firing the excimer laser. Set the preset laser power between 50-100 mJ/pulse on the laser control software itself, and set the preset repetition rate between 10-20 Hz in the Settings tab of the dosimetry software.
   1. Focus the laser beam using a plano convex lens mounted on a linear motorized stage. Measure the width and the height of the laser spot at the position of the capillary on a sticky note precisely using a caliper to calculate incident fluence (mJ/mm²) as shown in Figure 3B.
6. Place an opaque aperture near the capillary to ensure consistent illuminated width of the capillary regardless of changes in the beam size due to movement of the lens or changing the energy per pulse of the laser¹⁸.
   1. With the laser firing, move the motorized stage through its range of motion. Ensure that the beam stays centered on the aperture and the silhouette of the capillary can be observed throughout. The diameter of the aperture must be smaller than the width of the impinging focused beam at every point in the range of the motorized stage.
7. Run water through the capillary at 20 µL/min for at least one minute to wash the capillary.
   1. Click the Start Data + AutoZero button on the dosimeter software to zero the dosimeter to water and begin data collection.
      NOTE: If the buffer system for FPOP has significant UV absorbance at 265 nm, the FPOP system should be zeroed on the buffer, not water.
8. Set the calculated flow rate on the syringe pump.
   1. The flow rate of the protein sample depends on the irradiated volume per shot (V_Irr), the number of laser shots per second (R), and the desired unirradiated exclusion volume fraction (F_Ex) to correct for laminar flow effects and sample diffusion (0.15-0.30 recommended)^2,19,20. Calculate the V_Irr (in µL) based on the inner diameter of the capillary in mm (d) and the width of the laser spot impinging upon the capillary (i.e., the width of the aperture) in mm (w) using the following equation:
      V_Irr = π(d/2)²w
   2. Calculate the desired flow rate (in µL/min) based on the following equation:
      Flow = 60R[V_Irr (1 + F_Ex)]

2. Preparation of the protein solution for FPOP

1. Prepare the protein in the two or more different conditions to be compared (e.g., ligand-bound and ligand-free; aggregate and monomer; alone and with a protein-protein binding partner; etc.) for detecting the conformation changes.
2. Set the total volume used for FPOP to fit the needs of the experiment. The minimum limit usually depends on the volume of the irradiation capillary and the material required for robust detection and relative quantification, and will vary depending largely on the LC-MS/MS system used and the post-labeling sample processing method. The total volume for FPOP solutions commonly used in our group is 20 µL after the addition of hydrogen peroxide. The final concentration of the protein is commonly 1-10 µM, with 17 mM glutamine (to limit the lifetime of the hydroxyl radical), 1 mM adenine (to act as a radical dosimeter)^13,17 and 10 mM phosphate buffer (a buffer that is a poor scavenger of hydroxyl radicals). Samples are generally prepared with multiple replicates to allow for statistical modeling of results.
   1. For most general purposes, prepare samples in triplicate in both states, plus at least one sample to use as a no-laser control to measure background oxidation. Prepare 18 µL of this FPOP solution mix.
      NOTE: Many buffers and additives commonly used in biochemistry are hydroxyl radical scavengers. These additives and buffers can be used; however, reductions in oxidation due to hydroxyl radical scavenging of the buffer can occur. In general, keep all additives to the minimum required by the biological system to maximize protein oxidation yield. Dimethyl sulfoxide should be avoided due to the propensity to generate secondary radicals; dimethylformamide has been a useful alternative in our hands. When using buffers that are strong hydroxyl radical scavengers, glutamine can often be excluded from the FPOP solution mix.
3. Prepare 1 M hydrogen peroxide immediately before the FPOP experiment.
   NOTE: 30% hydrogen peroxide as commonly sold by vendors includes a stabilizer, which increases the shelf life. Once diluted, hydrogen peroxide should be used quickly, definitely within the same day. Hydrogen peroxide should also be regularly tested for decomposition by FPOP using a hydroxyl radical dosimeter.
4. Prepare microcentrifuge tubes containing 25 µL of quench solution of 0.5 µg/µL of methionine amide and 0.5 µg/µL catalase. If a sample volume greater than 20 µL is used for FPOP, increase the quench solution volume proportionally.

3. Perform the FPOP experiment

1. Add 2 µL of hydrogen peroxide in the 18 µL of the FPOP solution mix. Mix the contents gently with a pipette and quickly spin down the solution to the bottom of the microcentrifuge tubes. Immediately collect with a gastight syringe and load into the syringe pump.
2. Start the flow on the syringe pump with the flow rate as determined in step 1.8.1 (typically between 8-16 µL/min) by clicking the Start Pump button on the dosimeter software.
3. Monitor the real-time adenine reading using the inline dosimeter (see Table of Materials) and collect the sample in waste. Wait for the Abs₂₆₅ signal to stabilize.
4. Click on the Start Flash button in the dosimeter software to start firing the laser at the preset repetition rate and energy.
5. Monitor the real-time adenine reading using an inline dosimeter (see Table of Materials); the difference in Abs₂₆₅ with the laser off and the laser on is the ΔAbs₂₆₅ reading.
   NOTE: The appearance of highly unstable Abs₂₆₅ readings upon firing the laser in the presence of hydrogen peroxide is due to the generation of bubbles in solution. Reduce the fluence of the laser and/or the concentration of hydrogen peroxide to eliminate the bubbles.

4. Perform Compensation

NOTE: Different ligands, buffers, etc. may have different scavenging capacity towards hydroxyl radicals. It is important to ensure that comparable effective hydroxyl radical doses are available to react with protein across different samples. This is accomplished by ensuring equal hydroxyl radical dosimeter response between samples. Using adenine dosimetry, the change in UV absorbance at 265 nm (ΔAbs₂₆₅) reflects the effective hydroxyl radical dose; the larger the ΔAbs₂₆₅, the higher the effective hydroxyl radical dose.

1. Compare the ΔAbs₂₆₅ reading obtained with the inline dosimeter with the desired ΔAbs₂₆₅ reading obtained by prior experiments or controls. A ΔAbs₂₆₅ reading lower than the desired reading indicates an insufficient effective dose of hydroxyl radicals; a ΔAbs₂₆₅ reading indicates an effective radical dose that is too high. If the ΔAbs₂₆₅ reading is at the desired level, collect the sample immediately after laser irradiation in the quench buffer¹⁷.
2. Compensate the effective radical dose to equalize the ΔAbs₂₆₅. This compensation can be performed in three ways: change hydrogen peroxide concentration, increase laser fluence by changing the laser energy per pulse, or increase laser fluence by changing the focal plane of the focusing lens.
   1. To make a large change (>10 mAU) in ΔAbs₂₆₅ reading, remake the sample with more or less hydrogen peroxide and rerun the sample as per Section 3.
   2. To make a small change in ΔAbs₂₆₅ reading in real-time, adjust the focal plane of the incident beam by adjusting the position of the focusing lens using the 50 mm Motorized Stage. Bringing the focal plane closer to the position of the capillary will increase the ΔAbs₂₆₅ reading; bringing the focal plane farther from the position of the capillary will decrease the ΔAbs₂₆₅ reading.
3. Monitor the adenine ΔAbs₂₆₅ to measure the effective amount of hydroxyl radical present in the sample after laser irradiation¹³. Real-time monitoring with an inline UV capillary detector allows for real-time compensation as described in 4.2.2; adjust the lens position using the motorized stage until the ΔAbs₂₆₅ reading is equal to the desired reading. Post-experimental absorbance measurements with a UV spectrophotometer are also accurate, but require new samples to be used for each effective radical dose.

5. Digest the protein samples

NOTE: Trypsin is most commonly used to digest protein samples for FPOP, and is the protease used in this protocol. It is a reliable protease that generates peptides with basic sites both at the N- and C-terminus, promoting multiply charged peptide ions in MS. Moreover, it cleaves after lysine and arginine, two amino acids that are only moderately reactive to hydroxyl radicals; therefore, changes in the digestion pattern due to analyte oxidation is rare. Other proteases have been successfully used with FPOP²¹, but care should be taken to ensure digestion patterns are comparable between unoxidized and oxidized samples.

1. Measure the final volume of quenched FPOP sample. Add 500 mM Tris, pH 8.0 with 10 mM CaCl₂ containing 50 mM dithiothreitol (DTT) to the protein solution after quenching to a final concentration of 50 mM Tris, 1 mM CaCl₂ and 5 mM DTT.
2. Heat the protein sample at 95 °C for 15 minutes.
3. Immediately cool the sample on ice for 2 min.
4. Add the 1:20 trypsin/protein weight ratio to the samples.
5. Digest the protein overnight at 37 °C with mixing.
6. Stop the digestion reaction by the addition of 0.1% formic acid and/or heating the sample to 95 °C for 10 min.
7. Add 2 mM DTT to the samples and heat at 60 °C for 15 min immediately before LC-MS/MS.
   NOTE: While other groups have reported alkylation of thiols in FPOP experiments, in our hands we have noted side products upon alkylation of oxidized proteins (possibly due to reaction with nucleophilic carbonyls formed as a minor oxidation product). Therefore, we choose to avoid alkylation of thiols when possible.

6. Perform liquid chromatography-tandem mass spectrometry (LC-MS/MS)

1. Prepare the mobile phase A consisting of water containing 0.1% formic acid and mobile phase B consisting of acetonitrile with 0.1% formic acid.
2. Load the sample first onto a C18 trap column (300 µm I.D. x 5 mm 100 Å pore size, 5 µm particle size) trapping cartridge and wash with 2% solvent B for 3 minutes at a flow rate of 5.0 µL/min to remove salts and hydrophilic small molecules.
3. Then separate the peptides on C18 nanocolumn (0.75 mm x 150 mm, 2 µm particle size, 100 Å pore size) at a flow rate of 300 nL/min. The gradient consists of a linear increase from 2 to 35% solvent B over 22 min, ramped to 95% solvent B over 5 min and held for 3 min to wash the column, and then returned to 2% B over 3 min and held for 9 min to re-equilibrate the column.
   NOTE: This gradient is sufficient for LC-MS/MS of most one- and two-protein FPOP mixtures seeking to do peptide-level quantification. The percent of solvent B may need to be altered to increase peptide resolution in rare cases where peptides interfere with one another due to similar retention times and m/z values. Proteome-scale FPOP²² or experimental designs seeking to separate peptide oxidation product isomers^1,23,24,25 may require longer LC gradients and are beyond the scope of this report.
4. Elute the peptides directly into the nanospray source of a high-resolution mass spectrometer using a conductive nanospray emitter.
5. Acquire the data in positive ion mode. Set the spray voltage to 2400 V, and the temperature of the ion transfer tube to 300 °C.
6. Acquire the full MS scans from m/z 250 to 2000 at a nominal resolution at m/z 200 of 60,000 followed by eight subsequent data-dependent linear ion trap MS/MS scans on the top eight most abundant peptide ions using collision-induced dissociation at 35% normalized energy to identify the peptides. Fragment the peptides up to five times within 30 s and then transfer to an exclusion list for 60 s.

7. Data processing and calculation of average oxidation of peptides

1. Determine the sequence coverage of the protein, m/z values, and retention times of unoxidized peptides using the MS/MS proteomics search engine.
2. Set the precursor mass tolerance to 10 ppm and allow up to two missed cleavage sites for the trypsin digested samples, using standard trypsin cleavage specificity.
3. Set the peptide mass fragment mass tolerance to 0.4 Daltons.
4. Based on the m/z ratio of the unmodified peptides detected and the known mass shifts of the major oxidation products, calculate the m/z of the various theoretical oxidation products of each peptide^{4,26,27,28,29}.
5. Identify the extracted ion chromatogram of these m/z values using software to view the mass spectrometric run (Figure 4). Identify the peptide oxidation products based on their m/z, their charge state, and the similarity in elution time to the unmodified peptide. In our hands, peptide oxidation products elute between 240 seconds before to 180 seconds after the unmodified peptide using the LC gradient above. As oxidation will often result in multiple isomeric oxidation products, it is common to observe multiple partially resolved peaks in the extracted ion chromatograms of peptide oxidation products, as shown in Figure 4. Peptide oxidation products are quantified based on the area of the peak(s) in the extracted ion chromatograms.

Figure 4: Extracted ion chromatogram of a peptide and its oxidation products after FPOP. The m/z of the peptide oxidation products are calculated based on the m/z of the unoxidized peptide and the known oxidation products; and the areas of these peptide products are determined. The area of the peptide products is then used for the calculation of the average oxidation events per peptide. Please click here to view a larger version of this figure.

6. Calculate the average oxidation of the peptides using the following equation.

where P denotes the average number of oxidation events per peptide molecule, and I represents the peak area of the unoxidized peptide (Iunoxidized) and the peptide with n oxidation events. Note that I(singly oxidized) would include not only additions of a single oxygen atom but also other less-common single oxidation events that the investigator may choose to measure (e.g., oxidative decarboxylation, carbonyl formation, etc.)^{4,26,27,28,29}.