This work was funded by the National Institute of General Medical Sciences (R43GM125420 and R44GM125420).

1. Installing the capillary tube
<ol>
	<li>Using a silica cleaving stone, cleave 250 &#181;m inner diameter (ID) silica capillary to 27 inches. Check the capillary ends for a clean, straight cut.</li>
	<li>Create two windows roughly 15 mm in length by burning away the polyimide coating. Starting from the &#8220;lower end&#8221; make the first photolysis window 90 mm away from the &#8220;lower end&#8221; and the second dosimeter window 225 mm away from the &#8220;lower end&#8221;. 
	NOTE: Once the coating is burned away the capillary is very fragile.</li>
	<li>Unscrew the nut and ferrule at port 5 and insert the &#8220;lower end&#8221; of the capillary just beyond the conical end of the ferrule (Figure 1A).</li>
	<li>On the photolysis module, remove the photolysis cell cap by pulling it straight out and then remove the magnetically mounted metal mask which will hold the capillary in place. 
	CAUTION: Inside the photolysis cell cap is a curved mirror, do not allow anything to touch the mirror.</li>
	<li>On the dosimeter module, open the dosimeter cell by pushing on the tab on the left and swing the dosimeter cell open to the right. Remove the magnetically mounted clips which will hold the capillary in place.</li>
	<li>Place the capillary into the grooved channel in the base of the photolysis cell. Center the capillary window with the photolysis cell window. While holding the capillary in position with one hand, add the magnetic mask to hold the capillary in place. Place the photolysis cap back in position.</li>
	<li>Place the capillary into the grooved channel in the base of the dosimeter cell. Center the second capillary window on the small aperture at the center of the dosimeter cell base. While holding the capillary in position with one hand, place the two magnetic clips in position to hold the capillary in place. Close the dosimeter cell until it clicks closed.</li>
	<li>Insert the capillary through the knurled nut atop the capillary lift of the product collector (Figure 1B). Extend the capillary to just above the bottom of the vial. 
	NOTE: It is important for the capillary to reach the bottom of the vial to ensure that the capillary is submerged in the quench solution while running an experiment.</li>
</ol>
2. Installing an injection loop
<ol>
	<li>Use Teflon tubing with an outer diameter 1/16&#8221; and an inner diameter of 0.015&#8221; (381 &#181;m). Follow equation 1 to calculate the length of tubing needed for the desired volume using equation 1. 
	<img alt="Equation 1" src="/files/ftp_upload/61861/61861equ01.jpg" /> 
	Where L is the length of the tubing in millimeters, V is the desired volume in microliters and d is the tube inner diameter in millimeters. For an injection volume of 25 &#181;L and an inner diameter of 381 &#181;m, the tubing length needs to be roughly 219.3 mm. 
	NOTE: For volumes less than 20 &#181;L, use PTFE tubing with an inner diameter of 0.01&#8221;.</li>
	<li>Cut the Teflon tubing to the necessary length using a tube cutter. Check the ends of the tube for a clean, straight cut.</li>
	<li>Insert one end of the new injection loop through one of the nuts and place a new ferrule onto the end of the tube. Hold the ferrule and nut in place and insert the tube into port 3 of the injection valve until it bottoms out in the valve. Hold the tube firmly and tighten the nut finger tight. Using a wrench, tighten the nut &#188; turn further. Remove and inspect the assembly. 
	NOTE: If the ferrule is not fixed in position, reinstall and tighten 1/8 turn further. Repeat with the other end of the loop.</li>
	<li>Once both ends have a nut and fixed ferrule, loosely screw one end to port 3 and the other end to port 6 (Figure 1C). Once in position tighten both sides to finger tight then &#188; turn past finger tight with a wrench.</li>
</ol>
3. Initialize the photolysis system
<ol>
	<li>Turn on the photolysis modules in the following order: (1) Fluidics Module (2) Photolysis Module (3) Dosimetry Module (4) Product Collector, and lastly (5) the system computer and launch the control software 
	NOTE: Allow the Dosimeter Module at least a half-hour to warm up from a cold start.</li>
	<li>Fully submerge tubing from the &#8220;Valve&#8221; position on the syringe pump (Figure 1D) into 10 mL of buffer. Place the tubing from the &#8220;Waste&#8221; position (Figure 1D) and tubing from port 2 on the syringe port (Figure 1C) to an empty container to collect waste. 
	NOTE: Use the buffer your protein will be suspended in. A recommended buffer is 10 mM NaPO4.</li>
	<li>On the Product Collector carousel, place 1.5 mL microcentrifuge tubes at the position marked H and 6.</li>
	<li>If using 250 &#181;m ID capillary, set the load flow rate to 500 &#181;L/min, processing flow rate to 7.5 &#181;L/min, and waste flow rate to 500 &#181;L/min.</li>
	<li>Calculate the flash delay, product time, and waste time to use the Semi-Automatic Mode using equation 2. 
	<img alt="Equation 2" src="/files/ftp_upload/61861/61861equ02.jpg" /> 
	Where t is the time in minutes, s is the capillary distance in millimeters, d is the inner diameter of the capillary in millimeters, and f is the flow rate in &#181;L/min.
	<ol>
		<li>For the flash delay, the distance is from the &#8220;lower end&#8221; of the capillary to the first window, which should be roughly 90 mm. Using 250 &#181;m ID capillary and a processing flow rate of 7.5 &#181;L/min the sample will arrive to the photolysis window in about 35 seconds. Allow two flashes at 1 Hz to occur before the samples arrive, so set the flash delay to 33 seconds.</li>
		<li>For the product time, enter the amount of time injected solution will take to flow from the injection valve to the end of the capillary. For a 27&#8221; long 250 &#181;m ID capillary, set the product time to 4.5 minutes.</li>
		<li>For the waste time, enter the amount of time the total volume of injected solution will take to be collected. At this time, the product collector will move from the product position to the waste vial. For an injection volume of 25 &#181;L and a flow rate of 7.5 &#181;L/min, set the waste time to 7.8 minutes.</li>
	</ol>
	</li>
	<li>Rinse out the injection loop five times by injecting 25 &#181;L of HPLC-grade water into the injecting port with the injection valve set to the load position.</li>
	<li>Manually turn the injection valve up to &#8216;inject position&#8217; to flush the rest of the system. Select Process (Out) on the control software to begin flowing water. While flowing, raise the product collector capillary lift by selecting Up under the product collector so that you can see the end of the capillary. Flow water through the capillary until a droplet forms. 
	NOTE: If a droplet does not form, check the injector valve for leaks. If there is a leak, loosen the nut, reseat the capillary, and retighten.</li>
</ol>
4. Determine actual &#9642;OH yield to test radical scavenging effects from the buffer.
<ol>
	<li>In the Control Software, start the flow by selecting Process (Out). In the settings tab, set the flash voltage to 0 V. Under the Manual Control tab in the dosimeter data section, select Start Data + AutoZero.</li>
	<li>Select the position for the product vial (H) and waste vial (6).</li>
	<li>Select Ready, manually turn the injection valve down to the load position, and inject 25 &#181;L of 1 mM Adenine with 100 mM H2O2 into the injection port. Once injected, manually turn the injection valve up to the inject position. 
	NOTE: This automatically triggers the system to begin the flow, turn on the plasma source, and acquire the dosimetry data.</li>
	<li>Ramp the voltage up by navigating to the settings tab, and changing the flash voltage. Repeat steps 4.2 and 4.3 using 500 V, 750 V, 1000 V, and 1250 V. Perform the reading of adenine absorbance at each voltage in triplicates.</li>
	<li>Select calculations tab to determine the average absorbance of each sample. First, click Select, then manually select the beginning and end of the peak absorbance. In the available space, write in a description of the sample. Repeat for all acquired data. 
	NOTE: Bubbles may form causing a spike in dosimeter reading. When selecting data to determine average absorbance, omit areas with bubbles.</li>
	<li>Copy and paste data in Excel to calculate the average change in adenine absorbance for each voltage, thus determining the effective &#9642;OH yield.</li>
	<li>Repeat steps 4.1-4.6 if multiple sample conditions (different buffer/additives) are being used to normalize the effective &#9642;OH yield for each condition.</li>
</ol>
5. Modification of protein to detect changes in higher order structure.
<ol>
	<li>Mix 4 mM adenine with 20 &#181;M of protein in a one-to-one ratio to make a solution containing 2 mM adenine and 10 &#181;M protein.</li>
	<li>Make the quench solution using 0.3 mg/mL catalase to break down excess hydrogen peroxide and 35 mM methionine amide to quench any remaining radicals. Aliquot 25 &#181;L of quench solution into a 200 &#181;L microtube so that an equal volume of quench and the labeled product is mixed.</li>
	<li>Dilute H2O2 to 200 mM and keep on ice.</li>
	<li>On the Control Software in the settings tab, start the flash voltage at 0 V to determine any background oxidation.</li>
	<li>In the manual control tab, select Start Data + AutoZero, followed by Process (Out), then Ready, and finally turn the injection valve down to the load position.</li>
	<li>Place a quench solution in position 1 on the product collect carousel. On the System Control Software change the product vial to 1.</li>
	<li>Immediately before injection, mix 12.5 &#181;L of the adenine and protein mixture with 12.5 &#181;L of H2O2 to make a final concentration of 1 mM Adenine, 5 &#181;M protein, and 100 mM H2O2. Gently pipette up and down to mix, quickly spin down, and inject 25 &#181;L using the injection port within 10 seconds of mixing.</li>
	<li>Switch the injection valve to the inject position and wait while sample is being processed.</li>
	<li>Repeat acquisition with 500 V, 750 V, 1000 V, and 1250 V. Perform each voltage measurement in triplicate.</li>
	<li>Calculate the average absorbance as described in step 4.5. Copy and paste all data to Excel.</li>
</ol>
6. Shut the system down
<ol>
	<li>After all samples have been collected, flush out the syringe port and sample loop by setting the injection valve down to the load position and inject 25 &#181;L of HPLC water five times.</li>
	<li>Turn the injection valve up to the inject position to flush the rest of the system with HPLC water.</li>
	<li>Stop the flow, close the system control software, and turn off the modules starting with the product collector, dosimeter module, photolysis module, then finally the fluidics module.</li>
</ol>
7. Sample preparation and liquid chromatography-mass spectrometry
<ol>
	<li>Denature the protein by incubating samples at 80 &#176;C for 20 min in the presence of 50 mM Tris and 1 mM CaCl2. Cool samples to room temperature and add 1:20 trypsin to protein. Digest the protein overnight at 37 &#176;C with sample mixing. Next morning, terminate trypsin digestion by heating samples to 95 &#176;C for 10 minutes.</li>
	<li>Detect peptides using a high-resolution LC-MS/MS system connected with a UPLC system.</li>
	<li>Load the sample first onto trap column (300 &#181;m ID X 5 mm 100 &#197; pore size, 5 &#181;m particle size) and wash at 5.0 &#181;L/mL for 3 min with water containing 2% solvent B (acetonitrile and 0.1% formic acid).</li>
	<li>Separate the peptides on a C18 nanocolumn (0.75 mm x 150 mm, 2 &#181;m particle size, 100 &#197; pore size) at a flow rate of 300 nL/min with a gradient between solvent A (water containing 0.1% formic acid) and solvent B. The gradient for peptide elution consists of a linear increase from 2 to 35% B over 22 min, ramped to 95% 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.</li>
	<li>Acquire the data in positive ion mode. Set the spray voltage to 2400 V, and the temperature of the ion transfer tube to 300 &#176;C.</li>
	<li>Acquire the full MS scans from 250-2000 m/z followed by eight subsequent data-dependent MS/MS scans on the top eight most abundant peptide ions. Use collision-induced dissociation at 35% normalized energy to fragment the peptides.</li>
	<li>Identify all the unmodified peptides detected from MS/MS analysis using an available protein analysis software against the necessary FASTA file containing the protein sequence and the relevant proteolytic enzyme.</li>
	<li>Search and quantify modified peptides using a HRPF Data Processing Software. The extent of oxidation for each identified peptide is calculated by dividing the summed chromatographic peak area of a modified peptide by the total chromatographic peak area of that peptide modified and unmodified using equation 3. 
	P = [I(singly oxidized) X 1 + I (doubly oxidized) X 2 + I(triply oxidized) X 3 + .../[Iunoxidized + I(singly oxidized) + I(doubly oxidized) + I(triply oxidized) ...] 
	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.</li>
</ol>
8. For a differential study, repeat steps 5-7 on the second condition.
NOTE: To confirm reproducibility, two biological replicates in addition to technical triplicates for each condition are recommended.

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+photochemical+oxidation+of+proteins+for+epitope+mapping.">Fast photochemical oxidation of proteins for epitope mapping.</a> Analytical chemistry. 83 (20), 7657-7661 (2011).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+Energetic+Epitope+of+an+Antibody/Interleukin-23+Interaction+with+Hydrogen/Deuterium+Exchange,+Fast+Photochemical+Oxidation+of+Proteins+Mass+Spectrometry,+and+Alanine+Shave+Mutagenesis.">Mapping the Energetic Epitope of an Antibody/Interleukin-23 Interaction with Hydrogen/Deuterium Exchange, Fast Photochemical Oxidation of Proteins Mass Spectrometry, and Alanine Shave Mutagenesis.</a> Analytical chemistry. 89 (4), 2250-2258 (2017).</li><li>Liu, X. R., Zhang, M. M., Rempel, D. L., Gross, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Single+Approach+Reveals+the+Composite+Conformational+Changes,+Order+of+Binding,+and+Affinities+for+Calcium+Binding+to+Calmodulin.">A Single Approach Reveals the Composite Conformational Changes, Order of Binding, and Affinities for Calcium Binding to Calmodulin.</a> Analytical Chemistry. 91 (9), 5508-5512 (2019).</li><li>Kiselar, J. G., Janmey, P. A., Almo, S. C., Chance, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+analysis+of+gelsolin+using+synchrotron+protein+footprinting.">Structural analysis of gelsolin using synchrotron protein footprinting.</a> Molecular and Cellular Proteomics. 2 (10), 1120-1132 (2003).</li><li>Chea, E. E., Deredge, D. J., Jones, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+on+the+Conformational+Ensemble+of+Cyt+C+Reveal+a+Compact+State+during+Peroxidase+Activity.">Insights on the Conformational Ensemble of Cyt C Reveal a Compact State during Peroxidase Activity.</a> Biophysical Journal. 118 (1), 128-137 (2020).</li><li>Poor, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+paramyxovirus+fusion+(F)+protein-refolding+event+from+pre-+to+postfusion+by+oxidative+footprinting.">Probing the paramyxovirus fusion (F) protein-refolding event from pre- to postfusion by oxidative footprinting.</a> Proceedings of the National Academy of Sciences U S A. 111 (25), 2596-2605 (2014).</li><li>Roush, A. E., Riaz, M., Misra, S. K., Weinberger, S. R., Sharp, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+Buffer+Hydroxyl+Radical+Dosimetry+Using+Tris(hydroxymethyl)aminomethane.">Intrinsic Buffer Hydroxyl Radical Dosimetry Using Tris(hydroxymethyl)aminomethane.</a> Journal of American Society of Mass Spectrometry. 31 (2), 169-172 (2020).</li><li>Everett, E. A., Falick, A. M., Reich, N. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+critical+cysteine+in+EcoRI+DNA+methyltransferase+by+mass+spectrometry.">Identification of a critical cysteine in EcoRI DNA methyltransferase by mass spectrometry.</a> Journal of Biological Chemistry. 265 (29), 17713-17719 (1990).</li><li>Sanderson, R. J., Mosbaugh, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+specific+carboxyl+groups+on+uracil-DNA+glycosylase+inhibitor+protein+that+are+required+for+activity.">Identification of specific carboxyl groups on uracil-DNA glycosylase inhibitor protein that are required for activity.</a> Journal of Biological Chemistry. 271 (46), 29170-29181 (1996).</li></ol>

E.E.C., J.S.S., and S.R.W. disclose a significant financial interest in GenNext Technologies, Inc., an early stage company seeking to commercialize technologies for higher-order protein structure analysis. The representative data provided has been reviewed by S.K.M., who has no financial conflict of interest.

There are several critical steps to ensure proper labeling of proteins during any HRPF experiment. First, an appropriate flow rate and source flash rate are selected to make certain each bolus of the sample is irradiated once. This ensures that the protein is exposed to a single bolus of newly formed &#9642;OH. Once a protein is oxidized, the higher order protein structure can be altered. To be confident the native protein structure is probed, each protein molecule must be modified in a single instant. Dosimet...

Protein conformation and associated higher order structure (HOS) are the principal determinants of proper biological function and aberrant behavior1. The same applies to biopharmaceuticals, whose structure and functional activity is dependent on various aspects of their production and environment. Biopharmaceutical change in HOS have been linked to adverse drug reactions (ADR) attributed to undesirable pharmacology and patient immunological response2,3. The appearance of ADRs has alerted the biopharmaceutical industry to the critical role that protein HOS plays in the safety and efficacy of biotherapeutics, and they have established the need for new and improved HOS analytics4.
Hydroxyl Radical Protein Footprinting (HRPF) is a promising technique to track the change in protein HOS. HRPF involves the irreversible labeling of a protein&#8217;s exterior with &#9642;OH followed with mass spectrometry (MS) analysis to identify the solvent accessible surface of the protein5,6,7. HRPF has successfully been used to detect defects in protein HOS and its function8,9, characterize the HOS of monoclonal antibodies (mAb)10,11,12,13, determine the binding Kd of a ligand14, and much more15,16,17,18,19. A common method to generate the &#9642;OH for HRPF is Fast Photochemical Oxidation of Proteins (FPOP), which employs high-energy, fast UV lasers to produce &#9642;OH from photolysis of H2O2. For the most part, FPOP uses expensive excimer lasers employing hazardous gas (KrF) that demands substantial safeguards to avoid respiratory and eye injury20. To avoid inhalation hazards, others have used frequency quadrupled neodymium yttrium aluminum garnet (Nd:YAG) lasers21, which eliminates the use of toxic gas but are still costly, require significant operational expertise, and demand extensive stray light controls to protect users from eye injury.
Although ample information can be obtained using HRPF, broad adoption in biopharma has not been met. Two barriers for the limited HRPF adoption include: 1) the use of dangerous and expensive lasers that demand substantial safety precautions20; and 2) the irreproducibility of HRPF caused by background scavenging of &#9642;OH that limit comparative studies22. To supplant laser use, a high-speed, high energy plasma flash photolysis unit was developed to safely perform FPOP in a facile manner. To improve on the irreproducibility of HRPF experiments, real-time radical dosimetry is implemented.
The practice of HRPF has been limited by irreproducibility attributed to background scavenging of &#9642;OH22. While &#9642;OH are excellent probes of protein topography, they also react with many constituents found in preparations, making it necessary to measure the effective concentration of radical available to oxidize a target protein. Variations in buffer preparation, hydrogen peroxide concentration, ligand properties, or photolysis can result in oxidation differences between control and experimental groups that create ambiguity in HOS differential studies. The addition of real-time radical dosimetry enables the adjustment of the effect &#9642;OH load and therefore increases the confidence and reproducibility during an HRPF experiment. The use of radical dosimetry in FPOP has been described elsewhere23,24,25, and is further discussed in detail in a recent publication26. Here, we describe the use of a novel flash photolysis system and real-time dosimetry to label equine apo-myoglobin (aMb), comparing levels of peptide oxidation in an FPOP experiment to that of obtained when using an excimer laser.

<table>
	<tbody>
		<tr>
			<td>15 mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-53A</td>
			<td>any brand is sufficient</td>
		</tr>
		<tr>
			<td>50 &#181;L SGE Gastight Syringes</td>
			<td>Fisher Scientific</td>
			<td>SG-00723</td>
			<td></td>
		</tr>
		<tr>
			<td>Acclaim PepMap 100 C18 nanocolumn (0.75 mm X 150 mm, 2 &#181;m)</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile with 0.1% Formic Acid (v/v), LC/MS Grade</td>
			<td>Fisher Scientific</td>
			<td>LS120-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Apomyoglobin</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma-Aldrich</td>
			<td>C9322</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>022625501</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate Task Wipers</td>
			<td>Fisher Scientific</td>
			<td>06-666A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td>Fisher Scientific</td>
			<td>H325-100</td>
			<td>any 30% hydrogen peroxide is sufficient</td>
		</tr>
		<tr>
			<td>Methionine amide</td>
			<td>Chem-Impex</td>
			<td>03109</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002436</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbitrap Fusion Lumos Tribrid Mass Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>Orbitrap Fusion Lumos Tribrid Mass Spectrometer</td>
			<td>other high resolution instruments (e.g. Q exactive Orbitrap or Orbitrap Fusion) can be used</td>
		</tr>
		<tr>
			<td>Pierce Trypsin Protease, MS Grade</td>
			<td>Thermo Scientific</td>
			<td>90058</td>
			<td></td>
		</tr>
		<tr>
			<td>Polymicro Cleaving Stone, 1&#34; x 1&#34; x 1/32&#8221;</td>
			<td>Molex</td>
			<td>1068680064</td>
			<td>any capillary tubing cutter is sufficient</td>
		</tr>
		<tr>
			<td>UPLC</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water with 0.1% Formic Acid (v/v), LC/MS Grade</td>
			<td>Fisher Scientific</td>
			<td>LS118-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, LC/MS Grade</td>
			<td>Fisher Scientific</td>
			<td>W6-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser-free hydroxyl radical protein footprinting to perform higher order structural analysis of proteins

Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes in protein structure, protein-protein interactions, or protein-ligand interactions. HRPF utilizes hydroxyl radicals (&#9642;OH) to irreversibly label a protein&#8217;s solvent accessible surface. The inherent complexity, cost, and hazardous nature of performing HRPF have substantially limited broad-based adoption in biopharma. These factors include: 1) the use of complicated, dangerous, and expensive lasers that demand substantial safety precautions; and 2) the irreproducibility of HRPF caused by background scavenging of &#9642;OH that limit comparative studies. This publication provides a protocol for operation of a laser-free HRPF system. This laser-free HRPF system utilizes a high energy, high-pressure plasma light source flash oxidation technology with in-line radical dosimetry. The plasma light source is safer, easier to use, and more efficient in generating hydroxyl radicals than laser-based HRPF systems, and the in-line radical dosimeter increases the reproducibility of studies. Combined, the laser-free HRPF system addresses and surmounts the mentioned shortcomings and limitations of laser-based techniques.

The high-pressure plasma source coupled with real-time dosimetry allows better control of &#9642;OH yield to observe changes in higher-order protein structure more accurately. The addition of adenine allows for an effective real-time radical dosimeter. Upon oxidation, adenine loses UV absorbance at 265 nm (Figure 2A). The change in adenine absorbance is directly related to the concentration of radicals available for HRPF thus providing a means to effectively monitor changes in rad...

Watch this Scientific Journal Video about Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins at JoVE.com

Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins

This protocol presents a method to use inline radical dosimetry and a plasma light source to perform flash oxidation protein footprinting. This method replaces the hazardous UV laser to simplify and improve the reproducibility of fast photochemical oxidation of protein studies.

Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes ...

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3.5K Views.  GenNext Technologies Inc.. This protocol presents a method to use inline radical dosimetry and a plasma light source to perform flash oxidation protein footprinting. This method replaces the hazardous UV laser to simplify and improve the reproducibility of fast photochemical oxidation of protein studies.

Video: Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

This protocol describes the combination of size exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute characterization of proteins and complexes in solution. SEC-MALS determines the molecular weight and size of pure proteins, native oligomers, heterocomplexes and modified proteins such as glycoproteins.

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, and protein-protein interactions. FPOP utilizes a KrF excimer laser at 248 nm for photolysis of hydrogen peroxide to generate hydroxyl radicals which in turn oxidatively modify solvent-accessible amino acid side chains. Recently, we expanded the use of FPOP of in vivo oxidative labeling in Caenorhabditis elegans (C. elegans), entitled IV-FPOP. The transparent nematodes have been used as model systems for many human diseases. Structural studies in C. elegans by IV-FPOP is feasible because of the animal&#8217;s ability to uptake hydrogen peroxide, their transparency to laser irradiation at 248 nm, and the irreversible nature of the modification. The assembly of a microfluidic flow system for IV-FPOP labeling, IV-FPOP parameters, protein extraction, and LC-MS/MS optimized parameters are described herein.

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting technique that allows for mapping of protein structure in their native environment. This protocol describes the assembly and set-up of the IV-FPOP microfluidic flow system.

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. PRMTs can either mono- or dimethylate arginine guanidino groups symmetrically or asymmetrically. The enzymology of these proteins is a complex and intensely investigated area that requires milligram quantities of high-quality recombinant protein. The baculovirus expression vector system (BEVS) employing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda 9 (Sf9) insect cells has been used for expression screening and production of many PRMTs, including PRMT 1, 2, and 4 through 9. To simultaneously screen for the expression of multiple constructs of these proteins, including domains and truncated fragments as well as the full-length proteins, we have applied scalable methods utilizing adjustable and programmable multichannel pipettes, combined with 24- and 96-well plates and blocks. Overall, these method adjustments enabled a large-scale generation of bacmid DNA, recombinant viruses, and protein expression screening. Using culture vessels with a high-fill volume of Sf9 cell suspension helped to overcome space limitations in the production pipeline for single batch large-scale protein production. Here, we describe detailed protocols for the efficient and cost-effective expression of functional PRMTs for biochemical, biophysical, and structural studies.

The baculovirus expression vector system (BEVS) is a robust platform for expression screening and production of protein arginine methyltransferases (PRMTs) to be used for biochemical, biophysical, and structural studies. Milligram quantities of material can be produced for the majority of PRMTs and other proteins of interest requiring a eukaryotic expression platform.

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. ...

JUMPn: A Streamlined Application for Protein Co-Expression Clustering and Network Analysis in Proteomics

With recent advances in mass spectrometry-based proteomics technologies, deep profiling of hundreds of proteomes has become increasingly feasible. However, deriving biological insights from such valuable datasets is challenging. Here we introduce a systems biology-based software JUMPn, and its associated protocol to organize the proteome into protein co-expression clusters across samples and protein-protein interaction (PPI) networks connected by modules (e.g., protein complexes). Using the R/Shiny platform, the JUMPn software streamlines the analysis of co-expression clustering, pathway enrichment, and PPI module detection, with integrated data visualization and a user-friendly interface. The main steps of the protocol include installation of the JUMPn software, the definition of differentially expressed proteins or the (dys)regulated proteome, determination of meaningful co-expression clusters and PPI modules, and result visualization. While the protocol is demonstrated using an isobaric labeling-based proteome profile, JUMPn is generally applicable to a wide range of quantitative datasets (e.g., label-free proteomics). The JUMPn software and protocol thus provide a powerful tool to facilitate biological interpretation in quantitative proteomics.

We present a systems biology tool JUMPn to perform and visualize network analysis for quantitative proteomics data, with a detailed protocol including data pre-processing, co-expression clustering, pathway enrichment, and protein-protein interaction network analysis.