We acknowledge research funding from the National Institute of General Medical Sciences grant R43GM125420-01to support commercial development of a benchtop FPOP device and R01GM127267 for the development of standardization and dosimetry protocols for high-energy FPOP.

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.
<ol>
	<li>Prepare the FPOP optical bench.
	<ol>
		<li>Turn on the laser to warm up. Set ...

Joshua S. Sharp discloses a significant financial interest in GenNext Technologies, Inc., a small company seeking to commercialize technologies for protein higher order structure analysis including hydroxyl radical protein footprinting.

Mass spectrometry-based structural techniques, including hydrogen-deuterium exchange, chemical cross-linking, covalent labeling, and native spray mass spectrometry and ion mobility have been rapidly growing in popularity due to their flexibility, sensitivity, and ability to handle complex mixtures. FPOP boasts several advantages that has boosted its popularity in the area of mass spectrometry-based structural techniques. Like most covalent labeling strategies, it provides a stable chemical snapshot of protein topography ...

Fast photochemical oxidation of proteins (FPOP) is an emerging technique for the determination of protein topographical changes by ultra-fast covalent modification of the solvent-exposed surface area of proteins followed by detection by LC-MS1. FPOP generates a high concentration of hydroxyl radicals in situ by UV laser flash photolysis of hydrogen peroxide. These hydroxyl radicals are very reactive and short lived, consumed on roughly a microsecond timescale under FPOP conditions2. These hydroxyl radicals diffuse through water and oxidize various organic components in solution at kinetic rates generally ranging from fast (~106 M-1 s-1) to diffusion-controlled3. When the hydroxyl radical encounters a protein surface, the radical will oxidize the amino acid side chains on the protein surface, resulting in a mass shift of that amino acid (most commonly the net addition of one oxygen atom)4. The rate of the oxidation reaction at any amino acid depends on two factors: the inherent reactivity of that amino acid (which depends on the side chain and the sequence context)4,5 and the accessibility of that side chain to the diffusing hydroxyl radical, which closely correlates to the average solvent accessible surface area6,7. All of the standard amino acids except glycine have been observed as labeled by these highly reactive hydroxyl radicals in FPOP experiments, albeit at widely differing yields; in practice, Ser, Thr, Asn, and Ala are rarely seen as oxidized in most samples except under high radical doses and identified by careful and sensitive targeted ETD fragmentation8,9. After oxidation, samples are quenched to remove hydrogen peroxide and secondary oxidants (superoxide, singlet oxygen, peptidyl hydroperoxides, etc.) The quenched samples are then proteolytically digested to generate mixtures of oxidized peptides, where the structural information is frozen as a chemical &#8220;snapshot&#8221; in the patterns of oxidation products of the various peptides (Figure 1). Liquid chromatography coupled to mass spectrometry (LC-MS) is used to measure the amount of oxidation of amino acids in a given proteolytic peptide based on the relative intensities of the oxidized and unoxidized versions of that peptide. By comparing this oxidative footprint of the same protein obtained under different conformational conditions (e.g., ligand-bound versus ligand-free), differences in the amount of oxidation of a given region of the protein can be directly correlated with differences in the solvent-accessible surface area of that region6,7. The ability to provide protein topographical information makes FPOP an attractive technology for the higher-order structure determination of proteins, including in protein therapeutic discovery and development10,11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61580/61580fig01.jpg" /> 
Figure 1: Overview of FPOP.&#160;The surface of the protein is covalently modified by highly reactive hydroxyl radicals. The hydroxyl radicals will react with amino acid side chains of the protein at a rate that is strongly influenced by the solvent accessibility of the side chain. Topographical changes (for example, due to the binding of a ligand as shown above) will protect amino acids in the region of interaction from reacting with hydroxyl radicals, resulting in a decrease in the intensity of modified peptide in the LC-MS signal. <a href="https://www.jove.com/files/ftp_upload/61580/61580fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Different constituents present in the FPOP solution (e.g., ligands, excipients, buffers) have different scavenging activity towards the hydroxyl radicals generated upon the laser photolysis of hydrogen peroxide3. Similarly, a small change in peroxide concentration, laser fluence, and buffer composition may change the effective radical dose, making the reproduction of FPOP data challenging across the samples and between different labs. Therefore, it is important to be able to compare the hydroxyl radical dose available to react with protein in each sample using one of several available hydroxyl radical dosimeters12,13,14,15,16. Hydroxyl radical dosimeters act by competing with the analyte (and with all scavengers in solution) for the pool of hydroxyl radicals; the effective dose of hydroxyl radicals is measured by measuring the amount of oxidation of the dosimeter. Note that &#8220;effective hydroxyl radical dose&#8221; is a function of both the initial concentration of hydroxyl radical generated and the half-life of the radical. These two parameters are partially dependent on one another, making the theoretical kinetic modeling somewhat complex (Figure 2). Two samples could have wildly different initial radical half-lives while still maintaining the same effective radical dose by changing the initial concentration of hydroxyl radical formed; they will still generate identical footprints17. Adenine13 and Tris12 are convenient hydroxyl radical dosimeters because their level of oxidation can be measured by UV spectroscopy in real-time, allowing for researchers to quickly identify when there is a problem with effective hydroxyl radical dose and to troubleshoot their problem. To solve this issue, an inline dosimeter located in the flow system directly after the site of irradiation that can monitor the signal from adenine absorbance changes in real-time is important. This helps in carrying out FPOP experiments in buffers or any other excipient with widely differing levels of hydroxyl radical scavenging capacity17. This radical dosage compensation can be performed in real-time, yielding statistically indistinguishable results for the same conformer by adjusting the effective radical dose.
In this protocol, we have detailed procedures for performing a typical FPOP experiment with radical dosage compensation using adenine as an internal optical radical dosimeter. This method allows investigators to compare footprints across FPOP conditions that have different scavenging capacity by performing compensation in real-time.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61580/61580fig02.jpg" /> 
Figure 2: Kinetic simulation of dosimetry-based compensation.&#160;1 mM adenine dosimeter response is measured in 5 &#181;M lysozyme analyte with a 1 mM initial hydroxyl radical concentration (&#9642;OH t1/2=53 ns), and set as a target dosimeter response (black). Upon the addition of 1 mM of the scavenger excipient histidine, the dosimeter response (blue) decreases along with the amount of protein oxidation in a proportional manner (cyan). The half-life of the hydroxyl radical also decreases (&#9642;OH t1/2=39 ns). When the amount of hydroxyl radical generated is increased to give an equivalent yield of oxidized dosimeter in the sample with 1 mM histidine scavenger as achieved with 1 mM hydroxyl radical in the absence of scavenger (red), the amount of protein oxidation that occurs similarly becomes identical (magenta), while the hydroxyl radical half-life decreases even further (&#9642;OH t1/2=29 ns). Adapted with permission from Sharp J.S., Am Pharmaceut Rev 22, 50-55, 2019. <a href="https://www.jove.com/files/ftp_upload/61580/61580fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>Adenine</td>
			<td>Acros Organics</td>
			<td>147440250</td>
			<td>Soluble in water upto 3.5 mM</td>
		</tr>
		<tr>
			<td>Aperture</td>
			<td>Edmund Optics</td>
			<td>39-905</td>
			<td>1000 &#956;m Aperture Diameter, Gold-Plated Copper Aperture</td>
		</tr>
		<tr>
			<td>Aperture holder</td>
			<td>Edmund Optics</td>
			<td>53-287</td>
			<td>25.8mm Outer Diameter, Precision Pinhole Mount</td>
		</tr>
		<tr>
			<td>Catalse</td>
			<td>Sigma Aldrich</td>
			<td>C-40</td>
			<td>Catalase from bovine liver, lyophilized powder, &#8805;10,000 units/mg protein</td>
		</tr>
		<tr>
			<td>COMPex Pro laser</td>
			<td>Coherent</td>
			<td>1113836</td>
			<td>COMPexPRO 102, F-Vversion, KrF laser, No XeCl</td>
		</tr>
		<tr>
			<td>Dithiotheitol (DTT)</td>
			<td>Promega</td>
			<td>V3151</td>
			<td>DTT, Molecular Grade (DL-Dithiothreitol)</td>
		</tr>
		<tr>
			<td>Fraction collector</td>
			<td>GenNext Technologies, Inc.</td>
			<td>N/A</td>
			<td>Automated fraction collector</td>
		</tr>
		<tr>
			<td>Fused silica capillay</td>
			<td>Molex</td>
			<td>1068150023</td>
			<td>Polymicro Flexible Fused Silica Capillary Tubing, Inner Diameter 100 &#181;m, Outer Diameter 375 &#181;m, TSP100375</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Acros Organics</td>
			<td>119951000</td>
			<td>L(+)-Glutamine, 99%</td>
		</tr>
		<tr>
			<td>Holder for lens</td>
			<td>Edmund Optics</td>
			<td>03-668</td>
			<td>53 mm Outer Diameter, Three-Screw Adjustable Ring Mount</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Fisher Scientific</td>
			<td>H325-100</td>
			<td>Hydrogen Peroxide, 30% (Certified ACS), Fisher Chemical</td>
		</tr>
		<tr>
			<td>LC-MS/MS system</td>
			<td>Thermo Scientific</td>
			<td>IQLAAEGAAPFADBMBCX</td>
			<td>Dionex Ultimate 3000 coupled to Orbitap Fusion Tribrid mass spectrometer</td>
		</tr>
		<tr>
			<td>Mas spec grade Acetonitrile</td>
			<td>Fisher Scientific</td>
			<td>A955-1</td>
			<td>Acetonitrile, Optima LC/MS Grade, Fisher Chemical</td>
		</tr>
		<tr>
			<td>Mass spec grade formic acid</td>
			<td>Fisher Scientific</td>
			<td>A117-50</td>
			<td>Formic Acid, 99.0+%, Optima&#8482; LC/MS Grade, Fisher Chemical</td>
		</tr>
		<tr>
			<td>Mass spec grade water</td>
			<td>Fisher Scientific</td>
			<td>W6-4</td>
			<td>Water, Optima LC/MS Grade, Fisher Chemical</td>
		</tr>
		<tr>
			<td>MES buffer</td>
			<td>Sigma Aldrich</td>
			<td>M0164</td>
			<td>MES hemisodium salt</td>
		</tr>
		<tr>
			<td>Methionine amide</td>
			<td>Bachem</td>
			<td>4000594.0005</td>
			<td>H-met-NH2.HCl</td>
		</tr>
		<tr>
			<td>Micro V clamp</td>
			<td>Thor Labs</td>
			<td>VK250</td>
			<td>Micro V-clamp with stainless steel blades</td>
		</tr>
		<tr>
			<td>Motorized stage</td>
			<td>Edmund Optics</td>
			<td>68-638</td>
			<td>50mm Travel Motorized Stage System with Manual Control</td>
		</tr>
		<tr>
			<td>Nano C18 colum</td>
			<td>Thermo Scientific</td>
			<td>164534</td>
			<td>Acclaim PepMap 100 C18 HPLC Columns</td>
		</tr>
		<tr>
			<td>Optical bench</td>
			<td>Edmund Optics</td>
			<td>56-935</td>
			<td>18&#34; x 18&#34; breadboard</td>
		</tr>
		<tr>
			<td>Pioneer FPOP Module System</td>
			<td>GenNext Technologies, Inc.</td>
			<td>N/A</td>
			<td>Inline FPOP Radical Dosimetry System</td>
		</tr>
		<tr>
			<td>Post holder</td>
			<td>Edmund Optics</td>
			<td>58-979</td>
			<td>3&#34; Length, &#188;-20 Thread, Post Holder</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Fisher Scientific</td>
			<td>BP331-500</td>
			<td>Sodium Phosphate Dibasic Heptahydrate (Colorless-to-White Crystals), Fisher BioReagents</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Fisher Scientific</td>
			<td>BP330-500</td>
			<td>Sodium Phosphate Monobasic Monohydrate (Colorless-to-white Crystals), Fisher BioReagents</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Hamilton</td>
			<td>81065</td>
			<td>100 &#181;L, Model 1710 RN SYR, Small Removable NDL, 22s ga, 2 in, point style 3</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>KD Scientific</td>
			<td>788101</td>
			<td>Legato&#160;101 syringe pump</td>
		</tr>
		<tr>
			<td>Trap C18 column</td>
			<td>Thermo Scientific</td>
			<td>160454</td>
			<td>Thermo Scientific&#160;Acclaim PepMap 100 C18 HPLC Columns</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma Aldrich</td>
			<td>252859</td>
			<td>Tris(hydroxymethyl)aminomethane</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Promega</td>
			<td>V5111</td>
			<td>Sequencing Grade Modified Trypsin</td>
		</tr>
		<tr>
			<td>UV plano convex lens</td>
			<td>Edmund Optics</td>
			<td>84-285</td>
			<td>30 mm Dia. x 120 mm FL Uncoated, UV Plano-Convex Lens</td>
		</tr>
	</tbody>
</table>

enabling real-time compensation in fast photochemical oxidations of proteins for the determination of protein topography changes

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area of proteins. This technique relies on the reaction of amino acid side chains with hydroxyl radicals freely diffusing in solution. FPOP generates these radicals in situ by laser photolysis of hydrogen peroxide, creating a burst of hydroxyl radicals that is depleted on the order of a microsecond. When these hydroxyl radicals react with a solvent-accessible amino acid side chain, the reaction products exhibit a mass shift that can be measured and quantified by mass spectrometry. Since the rate of reaction of an amino acid depends in part on the average solvent accessible surface of that amino acid, measured changes in the amount of oxidation of a given region of a protein can be directly correlated to changes in the solvent accessibility of that region between different conformations (e.g., ligand-bound versus ligand-free, monomer vs. aggregate, etc.) FPOP has been applied in a number of problems in biology, including protein-protein interactions, protein conformational changes, and protein-ligand binding. As the available concentration of hydroxyl radicals varies based on many experimental conditions in the FPOP experiment, it is important to monitor the effective radical dose to which the protein analyte is exposed. This monitoring is efficiently achieved by incorporating an inline dosimeter to measure the signal from the FPOP reaction, with laser fluence adjusted in real-time to achieve the desired amount of oxidation. With this compensation, changes in protein topography reflecting conformational changes, ligand-binding surfaces, and/or protein-protein interaction interfaces can be determined in heterogeneous samples using relatively low sample amounts.

Comparison of the heavy chain peptide footprint of the adalimumab biosimilar in phosphate buffer and when heated at 55 &#176;C for 1 h show interesting results. Student&#8217;s t-test is used for the identification of peptides that are significantly changed in these two conditions (p &#8804; 0.05). The peptides 20-38, 99-125, 215-222, 223-252, 260-278, 376-413, and 414-420 show significant protection from solvent when the protein is heated to form aggregates (Figure 5)30</s...

Watch this Scientific Journal Video about Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes at JoVE.com

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

Fast photochemical oxidation of proteins is an emerging technique for the structural characterization of proteins. Different solvent additives and ligands have varied hydroxyl radical scavenging properties. To compare the protein structure in different conditions, real-time compensation of hydroxyl radicals generated in the reaction is required to normalize reaction conditions.

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area ...

The method facilitates measurement of protein confirmation and protein interactions. The buffer composition, sample purity and protein concentration requirements are flexible allowing the method to address challenging structural problems. This technique allows a compression of samples in a wide variety of different buffer systems.Using our dosimetry method, researchers can monitor how well their labeling reactions are working in real time. One application of this technology is in drug development because it allows researchers to test widely different drug formulations and how they affect protein structure. To prepare the FPOP optical bench.First turn on the laser and set the laser to external trigger, constant energy and no gas replacement. When the laser has warmed set the laser energy per pulse to between 80 to 120 milligrams per pulse and use a butane torch to gently burn the polyimide coding of the capillary at the spot at which the inline dosimeter will read the absorbent signal at 265 nanometers after laser exposure. Use a lint-free wipe and methanol to gently wipe the debris from the capillary and place the cleaned capillary through the beam path of the laser and into the inline dosimeter.Press the lever on the top of the inline dosimeter to open the hinge and remove the magnetic holders. Place the capillary in the machine groove of the inline dosimeter using the magnetic holders to keep the capillary in place and close the dosimeter hinge over the capillary pressing the hinge until the lever locks in place. Click the start flash button in the dosimetry software to begin firing the excimer laser and set the preset repetition rate between 10 to 20 Hertz in the dosimetry software with the laser firing move the motorized stage through its range of motion, ensuring that the beam stays centered on the aperture and that the silhouette of the capillary can be observed throughout.Then flush the capillary with water at 20 microliters per minute for at least one minute and click start data plus auto zero in the dosimeter software to zero the dosimeter to the water before beginning the data collection. To perform an FPOP experiment add two microliters of hydrogen peroxide to 18 microliters of FPOP solution and use a pipette to gently mix the solution quickly collect the solution at the bottom of the tube by centrifugation and immediately use a gas tight syringe to load the solution into a syringe pump. Click start pump and start data in the dosimeter software to start the flow on the syringe pump at the appropriate flow rate and use the inline dosimeter to monitor the real-time adenine reading until the absorbent signal at 265 nanometers stabilizes collecting the sample in a waste container.To start firing the laser at the preset repetition rate end energy click the start flash button in the dosimeter software and use the inline dosimeter to monitor the real-time adenine reading. The difference in the absorbance at 265 nanometers with the laser off and the laser on will be the delta absorbents at 265 nanometers reading. To ensure that comparable hydroxyl radicals are available to react with proteins across different samples.Compare the delta absorbents at 265 nanometers reading obtained with the inline dosimeter with the desire delta absorbents at 265 nanometers reading obtained from previous experiments or controls. If the reading is at the desired level collect the sample immediately after laser or radiation in the quench buffer. To compensate the effective radical dose to equalize the absorbance readings change the hydrogen peroxide concentration, change the laser energy per pulse or change the focal plane of the focusing lens to increase the laser fluence.To make a greater than 10 milli absorbance unit change in the delta absorbents reading remake the sample with more or less hydrogen peroxide and rerun the sample as demonstrated. To make a small change in the absorbance reading in real time, use the 50 millimeter motorized stage to adjust the position of the focusing lens to adjust the focal plane of the incident beam. To measure the effective amount of hydroxyl radical present in the sample after laser radiation monitor the adenine delta absorbents at 265 nanometers in real time with an inline UV capillary detector using the motorized stage to adjust the lens position until the delta absorbents at 265 nanometers reading is equal to the desired reading.Comparison of the heavy chain peptide footprint of the adilumumab biosimilar in phosphate buffer and after heating at 55 degrees Celsius for one hour reveals that the indicated peptide regions demonstrate significant protection from solvents when the protein is heated to form aggregates. Using an inline dosimeter in real time to monitor the difference in adenine absorbents before and after laser or radiation indicates that a comparable change in the adenine absorbents level is observed in MES buffer compared to phosphate buffer. While the average oxidation of the peptides is lower in the presence of MES buffer compared to phosphate buffer.However, as the laser fluence is increased to have an equal adenine dosimetry response the average peptide oxidation values are almost the same after FPOP and MES buffer and phosphate buffer. As the dosimetry reading is affected by the laser fluence peroxide concentration as well as the radical scavenger. Take care that the dosimetry response between the sample survey close to each other.Site directed mutagenesis is often used to probe sites protected in FPOP. This allows researchers to determine if changes are due to Alistair. This technique facilitates the comparison of proteins in different buffers or formulations allowing researchers to determine the effect of different additives on protein confirmation.

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JoVE Journal

Biochemistry

5.1K Görüntüleme.  University of Mississippi. Yöntem protein onayı ve protein etkileşimlerinin ölçülmesini kolaylaştırır. Tampon bileşimi, örnek saflık ve protein konsantrasyonu gereksinimleri, yöntemin zorlu yapısal sorunları ele almasına olanak tanıyan esnektir. Bu teknik, çok çeşitli farklı arabellek sistemlerinde numunelerin sıkıştırılamasına olanak sağlar.Dozimetri yöntemimizi kullanarak, araştırmacılar etiketleme reaksiyonlarının gerçek zamanlı olarak ne kadar iyi çalıştığını izleye...