Name: laser-free hydroxyl radical protein footprinting to perform higher order structural analysis of proteins
Uploaded: 2021-06-04T13:00:00
Description: Watch this Scientific Journal Video about Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins at JoVE.com

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...

<ol>
	<li>Nagarkar, R. P., Murphy, B. M., Yu, X., Manning, M. C., Al-Azzam, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+protein+higher+order+structure+using+vibrational+circular+dichroism+spectroscopy.">Characterization of protein higher order structure using vibrational circular dichroism spectroscopy.</a> Current Pharmaceutical Biotechnology. 14 (2), 199-208 (2013).</li><li>Giezen, T. J., Schneider, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+assessment+of+biosimilars+in+Europe:+a+regulatory+perspective.">Safety assessment of biosimilars in Europe: a regulatory perspective.</a> Generics and Biosimilars Initiative Journal. , 1-8 (2014).</li><li>Giezen, T. J., Mantel-Teeuwisse, A. 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M., 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=Complementary+MS+methods+assist+conformational+characterization+of+antibodies+with+altered+S-S+bonding+networks.">Complementary MS methods assist conformational characterization of antibodies with altered S-S bonding networks.</a> Journal of American Society of Mass Spectrometry. 24 (6), 835-845 (2013).</li><li>Storek, K. M., 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=Monoclonal+antibody+targeting+the+&#946;-barrel+assembly+machine+of+Escherichia+coli+is+bactericidal.">Monoclonal antibody targeting the &#946;-barrel assembly machine of Escherichia coli is bactericidal.</a> Proceedings of the National Academy of Sciences. , (2018).</li><li>Vij, R., 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=A+targeted+boost-and-sort+immunization+strategy+using+Escherichia+coli+BamA+identifies+rare+growth+inhibitory+antibodies.">A targeted boost-and-sort immunization strategy using Escherichia coli BamA identifies rare growth inhibitory antibodies.</a> Scientific Reports. 8 (1), 7136 (2018).</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>Lu, Y., 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=Fast+Photochemical+Oxidation+of+Proteins+Maps+the+Topology+of+Intrinsic+Membrane+Proteins:+Light-Harvesting+Complex+2+in+a+Nanodisc.">Fast Photochemical Oxidation of Proteins Maps the Topology of Intrinsic Membrane Proteins: Light-Harvesting Complex 2 in a Nanodisc.</a> Analytical Chemistry. 88 (17), 8827-8834 (2016).</li><li>Marty, M., Zhang, H., Cui, W., Gross, M., Sligar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpretation+and+Deconvolution+of+Nanodisc+Native+Mass+Spectra.">Interpretation and Deconvolution of Nanodisc Native Mass Spectra.</a> Journal of American Society of Mass Spectrometry. 25, (2013).</li><li>Johnson, D. T., Di Stefano, L. H., 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=Fast+photochemical+oxidation+of+proteins(FPOP):+A+powerful+mass+spectrometry+based+structural+proteomics+tool.">Fast photochemical oxidation of proteins(FPOP): A powerful mass spectrometry based structural proteomics tool.</a> Journal of Biological Chemistry. , (2019).</li><li>Chea, E. E., 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=Analyzing+the+structure+of+macromolecules+in+their+native+cellular+environment+using+hydroxyl+radical+footprinting.">Analyzing the structure of macromolecules in their native cellular environment using hydroxyl radical footprinting.</a> Analyst. 143 (4), 798-807 (2018).</li><li>Aprahamian, M. L., Chea, E. E., Jones, L. M., Lindert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rosetta+Protein+Structure+Prediction+from+Hydroxyl+Radical+Protein+Footprinting+Mass+Spectrometry+Data.">Rosetta Protein Structure Prediction from Hydroxyl Radical Protein Footprinting Mass Spectrometry Data.</a> Analytical chemistry. 90 (12), 7721-7729 (2018).</li><li>Linde. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Linde Specialty Gases of North America. , (2009).</li><li>Aye, T. T., Low, T. Y., Sze, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanosecond+laser-induced+photochemical+oxidation+method+for+protein+surface+mapping+with+mass+spectrometry.">Nanosecond laser-induced photochemical oxidation method for protein surface mapping with mass spectrometry.</a> Analytical Chemistry. 77 (18), 5814-5822 (2005).</li><li>Niu, B., Zhang, H., Giblin, D., 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=Dosimetry+determines+the+initial+OH+radical+concentration+in+fast+photochemical+oxidation+of+proteins+(FPOP).">Dosimetry determines the initial OH radical concentration in fast photochemical oxidation of proteins (FPOP).</a> Journal of American Society of Mass Spectrometry. 26 (5), 843-846 (2015).</li><li>Misra, S. K., Orlando, R., 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=Compensated+Hydroxyl+Radical+Protein+Footprinting+Measures+Buffer+and+Excipient+Effects+on+Conformation+and+Aggregation+in+an+Adalimumab+Biosimilar.">Compensated Hydroxyl Radical Protein Footprinting Measures Buffer and Excipient Effects on Conformation and Aggregation in an Adalimumab Biosimilar.</a> American Association of Pharmaceutical Scientists Journal. 21 (5), 87 (2019).</li><li>Olson, L. J., Misra, S. K., Ishihara, M., Battaile, K. P., Grant, O. C., Sood, A., Woods, R. J., Kim, J. P., Tiemeyer, M., Ren, G., Sharp, J. S., Dahms, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+regulation+of+lysosomal+enzyme+recognition+by+the+cation-independent+mannose+6-phosphate+receptor.">Allosteric regulation of lysosomal enzyme recognition by the cation-independent mannose 6-phosphate receptor.</a> Communications Biology. 3 (1), 498 (2020).</li><li>Sharp, J. S., Misra, S. K., Persoff, J. J., Egan, R. W., Weinberger, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+Time+Normalization+of+Fast+Photochemical+Oxidation+of+Proteins+Experiments+by+Inline+Adenine+Radical+Dosimetry.">Real Time Normalization of Fast Photochemical Oxidation of Proteins Experiments by Inline Adenine Radical Dosimetry.</a> Analytical Chemistry. 90 (21), 12625-12630 (2018).</li><li>Misra, S. K., 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=Enabling+Real-Time+Compensation+in+Fast+Photochemical+Oxidations+of+Proteins+for+the+Determination+of+Protein+Topography+Changes.">Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes.</a> Journal of Visualized Experiments. 163, (2020).</li><li>Hambly, D. M., 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=Laser+flash+photolysis+of+hydrogen+peroxide+to+oxidize+protein+solvent-accessible+residues+on+the+microsecond+timescale.">Laser flash photolysis of hydrogen peroxide to oxidize protein solvent-accessible residues on the microsecond timescale.</a> Journal of American Society of Mass Spectrometry. 16 (12), 2057-2063 (2005).</li><li>Liu, X. R., Zhang, M. M., 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=Mass+Spectrometry-Based+Protein+Footprinting+for+Higher-Order+Structure+Analysis:+Fundamentals+and+Applications.">Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications.</a> Chemical Reviews. 120 (10), 4355 (2020).</li><li>Jones, L. M., Sperry, B. J., Carroll, A. J., 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=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. 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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...

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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 ...

Laser-free hydroxyl radical protein footprinting makes identifying protein interaction sites and regions of confirmational change much easier, accelerating research in protein aggregation, structure, and stability studies. With inline radical dosimetry, users can adjust the effective hydroxyl radical load in real time, and with real time radical dosimetry, you can save experimental time and precious sample while improving labeling reproducibility. To begin, cleave a 250 micrometer inner diameter silica capillary to 27 inches with a silica cleaving stone, checking the ends of the capillary for a clean and straight cut.Create two windows of roughly 15 millimeter length. From the lower end of the capillary, burn away the polyimide coating 90 millimeters up to create the photolysis window and 225 millimeters for the dosimeter window. Insert the lower end of the capillary just beyond the conical end of a nut and ferrule.Add the capillary to port five, tightening just beyond finger tight with a wrench. Remove the photolysis cell cap of the photolysis module by pulling it straight out, then remove the magnetically mounted metal mask, which will hold the capillary in place. 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 that will hold the capillary in place. Then place the capillary into the grooved channel at the base of the photolysis cell, and center the capillary window with the photolysis cell window. Hold the capillary in position and add the magnetic mask.Place the photolysis cap back in position. Place the capillary into the grooved channel at the base of the dosimeter cell, and center the second capillary window on the small aperture at the center of the dosimeter cell. 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. Finally, insert the capillary through the knurled knob atop the capillary lift of the product collector, extending the capillary to just above the bottom of the vial. Cut the desired length of Teflon tubing using a cutter and check the ends for a clean straight cut.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 nut and ferrule in place while inserting the tube in port six of the injection valve until it bottoms out. Tighten the nut with a wrench, then confirm that the nut and ferrule are securely in place.Once both ends have a nut and fixed ferrule, loosely screw one end to port three and the other end to port six. Once in position, tighten both sides to finger tight, then a quarter turn past finger tight with a wrench. Turn on the laser-free HRPF system by starting the fluidics module, followed by the photolysis module, dosimeter module, product collector, and the system computer.Launch the system software. Fully submerge the tubing into 10 milliliters of buffer from the valve position on the syringe pump. Direct the tubing from the waste position and the tubing from port two on the syringe port to an empty container to collect waste.Place 1.5 milliliter microcentrifuge tubes at the position marked HN6 on the product collector carousel. Rinse out the injection loop five times by injecting 25 microliters of HPLC grade water with the valve set to the load position. Manually turn the injection valve to flush the rest of the system.Select Process on the control software to begin flowing water until a droplet forms. Make a solution containing two millimolar adenine and 10 micromolar protein. Then make a quench solution using 0.3 milligrams per milliliter catalase and 35 millimolar methianine amide.Aliquot 25 microliters of quench solution into a 200 microliter micro tube. Dilute hydrogen peroxide to 200 millimolar, keeping it on ice. Start the flash voltage at zero volts on the control software.This zero volt control will determine any background oxidation on the protein of interest. Select Start Data AutoZero, followed by Process then Ready. Finally, turn the injection valve down to the load position.Place the quench solution in position one on the product collector carousel, then change the product vial to one on the system control software. Immediately before injection, mix 12.5 microliters of the adenine and protein mixture with 12.5 microliters of hydrogen peroxide. Pipette the mixture up and down to mix, then quickly spin it down.Inject 25 microliters of this solution using the injection port within 10 seconds of mixing. Switch the injection valve to the inject position and wait while the sample is being processed. Turn up the flash voltage to 750 volts on the control software, and repeat the labeling steps.Then record the 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 this for all acquired data. Copy and paste the data into a spreadsheet to calculate the average change in adenine absorbance for each voltage. 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 microliters of HPLC water five times.Turn the injection valve up to the inject position to flush the rest of the system with HPLC water. Once flushed, stop the flow, exit the system control software, and turn off the modules, starting with the product collector, dosimeter module, photolysis module, and finally the fluidics module. Upon oxidation, adenine used for dosimetry decreases in UV absorbance at 265 nanometers.Buffer 2 contains a radical scavenger, which decreases the change in adenine absorbance compared to Buffer 1. Apomyoglobin was modified in the presence of hydrogen peroxide and adenine at four increasing plasma lamp voltages. Six peptides were detected, with a linear increase in oxidation with respect to the change of adenine absorbance at 265 nanometers.These six oxidized peptides are labeled on a crystal structure of myoglobin. The extent of oxidation detected from the high pressure plasma lamp is much higher than the laser-based method. This increase arises from the high pressure plasma lamp's broad spectrum UV emission spectrum.By producing a broad UV spectrum, the high pressure plasma lamp better overlaps the absorbance domain of hydrogen peroxide, resulting in a more efficient production of hydroxyl radicals through the photolysis of hydrogen peroxide, as compared to the Kr F Excimer Laser and Nd YAG Laser. The high pressure plasma lamp significantly increases the production of hydroxyl radicals beyond what is typically observed using a Kr F Excimer Laser. Using 100 millimolar peroxide can cause high levels of background oxidation.If the protein system is susceptible to peroxide-induced oxidation, decrease the concentration to as low as 10 millimolar. HRPF's irreversible label allows for downstream sample handling, including long protease digestions, you can add in tandem mass tag for multiplexing, and perform 2D chromatography, all improving peptide detection and concoordinate structural information.

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