It is important to note that this work was a collaborative effort between three institutions, two research groups, and one core facility. The distribution and workload were carried out as follows: Protein (Xrn-1) expression was carried out at the University of Denver (Denver, CO). Oligonucleotide synthesis, quantification, and experimentation (mainly enzymatic degradation) were conducted at the University of Colorado Denver (Denver, CO). Optimization was also carried out there. MALDI-TOF spotting, acquisition, and analysis were carried out at the Analytical Resources Core Facility at Colorado State University. (Fort Collins, CO). SS would like to acknowledge a UROP Award (CU Denver) and Eureca grants (CU Denver) for support. E. G. C. acknowledges support from NIGMS, via R00GM115757. MJER acknowledges support from NIGMS, via 1R15GM132816. K. B. acknowledges resource ID: SCR_021758. The work was also supported by a Teacher-Scholar Award (MJER), TH-21-028, from the Henry Dreyfus Foundation.

RNase-free ultra pure water (Table 1) was used for the present study.
1. Concentration determination of RNA solution
<ol>
	<li>Prepare the RNA sample following the steps below.
	<ol>
		<li>Use a microcentrifuge tube (0.6 mL) to prepare a solution of RNA by diluting 1 &#181;L of stock solution (obtained via solid-phase synthesis)19 into 159 &#181;L of RNase-free H2O. Mix the solution by pipetting the mixture up and down repeatedly (10x). 
		NOTE: Since a wide range of cuvettes are commercially available, the needed volumes may differ. Cuvettes with a 1 cm pathlength and reduced volume (150 &#181;L) were used in this work.</li>
		<li>Rinse the UV-vis cuvette with methanol (2x), followed by water (ultrapure H2O, 3x, see Table of Materials). Use a flow of nitrogen gas to dry the cuvette thoroughly.</li>
		<li>Use a pipette to transfer the solution (step 1.1.1) into the cuvette, ensuring no air bubbles. Cap the cuvette and place it to the side until needed.</li>
	</ol>
	</li>
	<li>Determine the concentration of the prepared RNA solution using UV-vis Spectrophotometer.
	<ol>
		<li>Turn on the UV-Vis spectrophotometer by flipping the switch located in the back of the instrument. Allow the instrument to complete the startup procedure and click on the UV WinLab icon to open the instrument operation/control window.</li>
		<li>Turn on the Peltier Temperature Controller spectrophotometer using the switch located to the instrument&#39;s right.</li>
		<li>Under Base Methods, click on Scan - Lambda 365. Another screen will open and prompt the user to ensure that the cell holders are empty. Click on OK and allow the instrument to conduct its system checks. The system will then display a list of checks, ensure that all &#34;Pass&#34; and click on OK. 
		NOTE: It is recommended to follow steps 1.2.1-1.2.3 in the provided order; altering this sequence of events may lead to software malfunction.</li>
		<li>Adjust the scanning parameters by selecting Data Collection. In the new window under Scan Settings change the Start to 350 nm and the End to 215 nm.</li>
		<li>Activate the Peltier by selecting the + to the left of the Accessory. Click on Multiple Cell Peltier, change the Temperature &#176;C to 25, and click on Peltier On.</li>
		<li>Navigate to the Sample Info tab and enter the number of samples, names, and cell position as desired.</li>
		<li>Click on Autozero and insert the cuvette containing the background solution. Ensure to position this cuvette in the same slot where measurements will be carried out.</li>
		<li>Click on Start and insert the cuvette (step 1.1.3) in the desired cell. Ensure that the cuvette window is oriented parallel to the front face of the instrument. Click on OK to begin the first scan at 25 &#176;C.</li>
		<li>For measurements at higher temperatures, repeat steps 1.2.5-1.2.8 and change the temperature to the desired sample temperature (90 &#176;C in the present case).</li>
		<li>Click on File &#62; Export and choose the desired file location. Select XY Data to obtain a .txt file. 
		NOTE: An illustrative aid of step 1.2 can be found in Supplementary File 1 (S1-S7).</li>
	</ol>
	</li>
	<li>Perform data acquisition for determining the RNA concentration.
	<ol>
		<li>Find the file for each spectrum under the Results tab. To obtain the absorbance, hover over the line, navigate to 260 nm, and record the displayed absorbances. Alternatively, through the File &#62; Export cascade, export the data as a .txt file for further graphical analysis using other plotting software. 
		NOTE: If 0.1 &#60; A &#8805; 1, it will be necessary to either dilute or increase the concentration of the RNA sample prepared in step 1.1.1.</li>
		<li>Use the Beer-Lambert law to calculate the concentration of the solution. 
		NOTE: Beer&#39;s Law: A = &#949;cl, where: 
		A:&#160;Absorbance (at 260 nm in this case, from step 1.3.1) 
		&#949;: Extinction coefficient. For the sequence used herein, 208,000 L mol-1cm-1 is the value obtained from an OligoAnalyzer tool. 
		l: Path length, 1 cm 
		c: concentration of the sample</li>
		<li>Use these calculations to prepare the desired solution of RNA to be used for subsequent experiments. Solutions with [RNA] = 200 &#181;M were used in the present study.</li>
	</ol>
	</li>
</ol>
2. Hydrolysis of oligonucleotides of RNA by Xrn-1
<ol>
	<li>Perform 5&#39; phosphorylation of RNA following the steps below.
	<ol>
		<li>Use a 0.6 mL microcentrifuge tube to prepare the following solution (50 &#181;L total volume).
		<ol>
			<li>Add RNase-free H2O (33.5 &#181;L); volumes may vary depending on [RNA].</li>
			<li>Then, add solution A (5 L, Table 1).</li>
			<li>Add an aqueous solution of adenosine triphosphate (ATP, see Table of Materials) (6 L, 10 mM, 60 nmol).</li>
			<li>Add RNA aqueous solution (1 &#181;L, 200 &#181;M, 200 pmol).</li>
			<li>Add polynucleotide kinase (PNK enzyme, see Table of Materials) solution (4.5 L, 45 units). Mix gently by pipetting the mixture up and down (10x).</li>
		</ol>
		</li>
		<li>Incubate the reaction mixture at 37 &#176;C for 45 min by placing it in a water bath.</li>
		<li>Inactivate the enzyme by placing the reaction tube in a pre-heated heat block (65 &#176;C) and incubating the solution for 10 min.</li>
		<li>Allow the solution to cool to room temperature (RNA phosphorylation was characterized via MALDI-TOF analysis, see Representative Results) to yield a final 5&#39;-phosphorylated RNA solution (4 &#181;M, 50 &#181;L).</li>
	</ol>
	</li>
	<li>Perform RNA hydrolysis by Xrn-1 following the steps below.
	<ol>
		<li>Using the solution (50 &#181;L) from step 2.1.4, carry out the following steps.
		<ol>
			<li>Add solution B (5 L, Table 1).</li>
			<li>Add a solution of Xrn-1 (0.5 L, 1 ng, 30 fmol). Mix the solution by gently pipetting up and down (10x).</li>
			<li>Incubate the reaction tube at room temperature for 2 h.</li>
		</ol>
		</li>
		<li>Transfer the reaction mixture (step 2.2.1.3) into a 10 kDa pore-sized centrifugal device (see Table of Materials), and filter the enzymes by centrifuging (700 x g for 10 min at room temperature).</li>
		<li>Transfer the filtrate into a 0.6 mL centrifuge tube.</li>
		<li>Wash the residual RNA on the centrifugal tube (step 2.2.2.) by adding RNase-free H2O (20 L), followed by centrifugation (700 x g for 10 min at room temperature).</li>
		<li>Combine the filtrate with that obtained from step 2.2.3.</li>
		<li>Store the tube containing the resulting solution in a freezer (0 &#176;C) or ship for analysis.</li>
	</ol>
	</li>
</ol>
3. Desalting of RNA solution, MALDI-TOF plate spotting
<ol>
	<li>Desalt and concentrate the sample using commercially available cation exchange C18 pipette tips (see Table of Materials).
	<ol>
		<li>Position a 10 L C18 pipette tip (pipette tip loaded with a bed of C18 chromatography media fixed at its end) onto a 10 &#181;L pipette, and then continue with the following:
		<ol>
			<li>Wash the C18 tip with 50% aqueous acetonitrile (10 &#181;L) solution two times. Discard the used volumes into a separate waste tube each time.</li>
			<li>Equilibrate the C18 tip with an aqueous trifluoroacetic acid solution (0.1% TFA, 10 &#181;L) two times. Discard the used volumes into a separate waste tube each time.</li>
			<li>Manually remove the C18 tip from the pipette and secure it onto a 200 &#181;L pipette containing a P200 pipette tip (the C18 tip will now be attached to the P200 pipette tip).</li>
			<li>Immerse the C18 tip into the solution (step 2.2.6), and aspirate-release the solution through the C18 tip ten times. 
			NOTE: The RNA sample becomes bound to the cation exchange resin inside the tip.</li>
			<li>Detach the C18 tip from the P200 pipette and position it onto a 10 &#181;L pipette.</li>
			<li>Wash the C18 tip using an aqueous solution of 0.1% TFA (10 &#181;L) two times. Discard the used volumes into a separate waste tube each time.</li>
			<li>Wash the C18 tip with RNase-free water (10 &#181;L) two times. Discard the used volumes into a separate waste tube each time.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Spot onto the MALDI plate following the steps below.
	<ol>
		<li>Elute the RNA oligonucleotide from the C18 tip (step 3.1.1.7) by immersing the sample into the desired matrix (10 &#181;L, 1:1 solution of C and D, Table 1), followed by dispensing the solution back into the tube. Repeat this process ten times.</li>
		<li>Pipette the solution from 3.2.1 onto two separate spots on the plate (1 &#181;L each, 20 pmol). Allow spots to air dry (Figure 1A and Video 1). 
		NOTE: This process can be repeated on the same spot to increase the sample concentration on each spot.</li>
		<li>Pipette desired calibrant (1 L) on two separate spots physically close to the sample location and allow to air dry. 
		NOTE: The test standard used in the present study was obtained from commercial sources (see Table of Materials).</li>
		<li>Over-lay the [fully dried] calibrant with the desired matrix (1 L). Allow to air dry. 
		&#8203;NOTE: It is important to track the position where the samples and calibrants are spotted prior to insertion into the instrument. Make a note of this using the plate coordinate system, e.g., sample 1 is located at position (C,3)</li>
	</ol>
	</li>
</ol>
4. Data acquisition and processing
NOTE: In general, THAP requires higher laser power to achieve ionization. Additionally, oligos can be difficult to ionize without fragmenting. Thus, it is usually necessary to use as low laser power as possible and raise detector gains and/or lower laser frequency to maximize detection and minimize fragmentation.
<ol>
	<li>Perform data acquisition following the steps below. 
	NOTE: Molecular weight measurements were performed using a mass spectrometer (see Table of Materials) in positive ion, linear mode with an ion source voltage of 20 kV, detector gain of ~2.8 kV, and a laser frequency of 20 Hz. Scan ranges were set based on expected molecular weight(s). These were calculated and expressed as mass over charge (m/z), where the charge was 1. External calibration was performed using a protein calibration mixture (Bacterial Test Standard, see Table of Materials) on a spot adjacent to the sample. The raw data was then processed in the flexAnalysis software (see Table of materials).
	<ol>
		<li>Open &#34;Flexcontrol software&#34; for operation. Press the instrument&#39;s green in/out button, wait for the target stage to move, and vacuum to vent. Open the lid, place the fully dried target plate into the instrument. Ensure to align in the right orientation (Figure 1B).</li>
		<li>Gently lower the lid, and then press the green in/out button. Wait for the plate to be retracted and the instrument to pump back down. Once the status bar (lower right corner of the software window) reflects &#39;Ready&#39;, proceed with calibration.</li>
		<li>To calibrate the instruments using the software, carry out the following.
		<ol>
			<li>Select the desired method by clicking on the File &#62; Select Method or pressing the Select button adjacent to the loaded method. Click on the Coordinate for the calibrant spot. Navigate to the Calibration tab and confirm the correct calibrant is selected. Ensure Random Walk is turned off (in the Sample Carrier tab).</li>
			<li>Press Start and manually direct the laser along a crystal (clicking the mouse arrow at the desired location in the camera view). Adjust laser power, if necessary. Once satisfied with the settings, press Start to collect spectra and add them to the sum buffer.</li>
			<li>Add spectra to the sum until sufficient intensity is reached. Toggle to view only the sum buffer, subtract the baseline, and smooth. Then click on Automatic Assign. Check each assigned peak and note the ppm error. If satisfied with assignments and errors, click on Apply.</li>
			<li>Clear the sum buffer and navigate to the position of the first sample.</li>
			<li>Confirm the desired scan range (&#39;Detection&#39; tab). Adjust the bars in the &#34;Mass Range&#34; so that the green region is representative of expected mass(es).</li>
			<li>Click directly on the Magnification window so that the crosshairs are oriented at the desired crystalized fragment (larger crystals often yield the best results, Figure 2).</li>
			<li>Collect a few spectra along the large crystals, adjusting the laser power as necessary. The proper setting is 5%-10% above the power at which peaks appear.</li>
		</ol>
		</li>
		<li>To obtain spectra, perform the following.
		<ol>
			<li>Click on Start to observe the laser flash in tandem with increasing spectral intensity (shown in the window in the upper right). Move the laser along the length of the crystal by clicking the mouse arrow up and down the crystal (Video 2).</li>
			<li>Click on Add directly below Start.</li>
			<li>Repeat this several times until desired intensity/number of shots is reached (reflected on the y-axis of the spectral window)</li>
			<li>Adjust the laser power and/or speed and/or detector gains if the initial scans are unsatisfactory. Repeat steps 4.1.4.1-4.1.4.3 as needed.</li>
			<li>If satisfied with the spectrum, click on Save As to save the spectrum under a specified name. Then, if other samples are needed, click on Clear Sum and repeat steps under step 4.1.4.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform data processing. 
	NOTE: Step 4.2 describes one way to analyze the data using the flexAnalysis software.
	<ol>
		<li>Open the desired software. Then, open spectrum (a) by selecting File. From the dropdown menu, select Open.</li>
		<li>A new window will be displayed. Click on Browse to locate the files saved from step 4.1.4. Check the boxes for the desired files, and then click on Open on the left. If only a single spectrum requires analysis, simply drag, and drop the file into the software window.</li>
		<li>Highlight the loaded file(s) for bulk analysis. Navigate to the Process tab on the toolbar. In the dropdown, select Subtract Mass Spectrum Baseline. 
		NOTE: The algorithm used for the subtraction will be determined by the method selected in step 4.1.3.</li>
		<li>Navigate to the Process tab on the toolbar. In the dropdown, select Smooth Mass Spectrum. 
		NOTE: The algorithm used for the smoothing will be determined by the method selected in step 4.1.3.</li>
		<li>Click on the Mass List tab; in the dropdown, select Find. This command automatically labels peaks with their masses; a list of the masses will be displayed on the screen to the right.</li>
		<li>To add or delete peaks, proceed to the Mass List tab. In the dropdown, click on Edit.</li>
		<li>Hover over the x-axis of the spectrum; a vertical line represents the location of the spectrum where the cursor is on. 
		NOTE: An icon with a graph and cursor will appear when a new mass can be added by clicking. This icon will appear when hovering over peaks that can be deleted by clicking (see step 4.2.7 in Supplementary File 1).</li>
		<li>Repeat steps 4.1.3-4.1.7 for additional spectra. Export the mass list for all spectra by clicking and dragging to highlighting the files on the left and execute the following menu cascade File &#62; Export &#62; Mass List to Excel.</li>
		<li>To export spectra as shown on the screen, either take a screenshot or export it as a report. For the latter, navigate to the Report menu. In the dropdown, select Save as PDF. 
		NOTE: One can change how the spectra are displayed using the three tabs below the spectra and/or varying the highlighted files. A screen will pop up. Name the PDF document as desired, adjust the settings/layout, and click on Save to generate an image of the spectrum.</li>
		<li>The image generated will reflect the spectral view in the software program. To manipulate the view, perform the following.
		<ol>
			<li>Zooming in: Click on the Zoom In button in the toolbar.</li>
			<li>Hover over the x-axis and click until the desired range is achieved.</li>
			<li>Zooming out/undo: Click on the Zoom Out button and click anywhere in the spectrum to return to full view. 
			NOTE: An illustrative aid for steps 4.1 and 4.2 can be found in Supplementary File 1 (pp S8-S23 and S24-S30, respectively).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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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=Biochemical+characterization+of+yeast+Xrn1.">Biochemical characterization of yeast Xrn1.</a> Biochemistry. 59 (15), 1493-1507 (2020).</li><li>Stevens, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+a+Saccharomyces+cerevisiae+exoribonuclease+which+yields+5'-mononucleotides+by+a+5'+leads+to+3'+mode+of+hydrolysis.">Purification and characterization of a Saccharomyces cerevisiae exoribonuclease which yields 5'-mononucleotides by a 5' leads to 3' mode of hydrolysis.</a> Journal of Biological Chemistry. 255 (7), 3080-3085 (1980).</li><li>Yan, L. L., Simms, C. L., McLoughlin, F., Vierstra, R. D., Zaher, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+and+alkylation+stresses+activate+ribosome-quality+control.">Oxidation and alkylation stresses activate ribosome-quality control.</a> Nature Communications. 10 (1), 5611 (2019).</li><li>Fasnacht, 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=Dynamic+23S+rRNA+modification+ho5C2501+benefits+Escherichia+coli+under+oxidative+stress.">Dynamic 23S rRNA modification ho5C2501 benefits Escherichia coli under oxidative stress.</a> Nucleic Acids Research. 50 (1), 473-489 (2022).</li><li>Estevez, M., Valesyan, S., Jora, M., Limbach, P. A., Addepalli, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+damage+to+RNA+is+altered+by+the+presence+of+interacting+proteins+or+modified+nucleosides.">Oxidative damage to RNA is altered by the presence of interacting proteins or modified nucleosides.</a> Frontiers in Molecular Biosciences. 8, 697149 (2021).</li><li>Tomar, 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=DNA+sequence+modulates+the+efficiency+of+NEIL1-catalyzed+excision+of+the+aflatoxin+B1-induced+formamidopyrimidine+guanine+adduct.">DNA sequence modulates the efficiency of NEIL1-catalyzed excision of the aflatoxin B1-induced formamidopyrimidine guanine adduct.</a> Journal of the American Chemical Society. 34 (3), 901-911 (2021).</li><li>Gaffney, 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=HIV-1+env-dependent+cell+killing+by+bifunctional+small-molecule/peptide+conjugates.">HIV-1 env-dependent cell killing by bifunctional small-molecule/peptide conjugates.</a> ACS Chemical Biology. 16 (1), 193-204 (2021).</li><li>Sikorski, E. L., 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=Selective+display+of+a+chemoattractant+agonist+on+cancer+cells+activates+the+formyl+peptide+receptor+1+on+immune+cells.">Selective display of a chemoattractant agonist on cancer cells activates the formyl peptide receptor 1 on immune cells.</a> ChemBioChem. , 202100521 (2022).</li><li>Kubo, T., Nishimura, Y., Sato, Y., Yanagihara, K., Seyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sixteen+different+types+of+lipid-conjugated+siRNAs+containing+saturated+and+unsaturated+fatty+Acids+and+exhibiting+enhanced+RNAi+potency.">Sixteen different types of lipid-conjugated siRNAs containing saturated and unsaturated fatty Acids and exhibiting enhanced RNAi potency.</a> ACS Chemical Biology. 16 (1), 150-164 (2021).</li></ol>

The authors have nothing to disclose.

The main challenge in this workflow arose between finalizing the experiments and carrying out the mass spectrometric analyses. Experiments were carried out and completed at the University of Colorado Denver and shipped (overnight) to the Colorado State University facilities. Data acquisition was carried out upon receipt, as per convenience. Several unexpected circumstances led to time delays in the process. In one instance, unexpected instrument malfunctions required the samples to be frozen (one time for 21 days) prior ...

MALDI-TOF1,2,3 is a widely used technique for the characterization and/or detection of molecules of varying sizes and characteristics. Some of its uses include diverse applications such as detecting tannins from natural resources4, imaging metabolites in food5, discovery or monitoring of cellular drug targets or markers6, and clinical diagnostics7, to name a few. Of relevance to the present work is the use of MALDI-TOF with DNA or RNA, with its use on oligonucleotides dating back to over three decades8, where several limitations were noted. This technique has now evolved to a reliable, commonly used means to characterize both biopolymers9 and identify/understand chemical and biochemical reactions, e.g., characterization of platinated sites in RNA10, identification of RNA fragments following strand cleavage11,12, or formation of protein-DNA cross-links13. Thus, it is valuable to illustrate and highlight important aspects of using this technique. The basics of MALDI-TOF have been described in video format as well14 and will not be further elaborated herein. Furthermore, its application in a DNA or protein context has been previously described and illustrated in the said format15,16,17.
The protocol for detecting RNA fragments formed after enzymatic hydrolysis is reported herein. The experimental model was chosen based on a recent finding published by our group18, where MALDI-TOF was used to determine the unique reactivity between the exoribonuclease Xrn-1 and oligonucleotides of RNA containing the oxidative lesion 8-oxoG. The 20-nucleotide long strands were obtained via solid-phase synthesis19, [5&#39;-CAU GAA ACA A(8-oxoG)G CUA AAA GU] and [5&#39;-CAU GAA ACA A(8-oxoG)(8-oxoG) CUA AAA GU], while Xrn-1 was expressed and purified following the previously described report20. In brief, Xrn-121 is a 5&#39;-3&#39; exoribonuclease with various key biological roles that degrade multiple types of RNA, including oxidized RNA22. It was found that the processivity of the enzyme stalls upon encountering 8-oxoG, which led to RNA fragments containing 5&#39;-phosphorylated ends [5&#39;-H2PO4-(8-oxoG)G CUA AAA GU], [5&#39;-H2PO4-(8-oxoG)(8-oxoG) CUA AAA GU], and [5&#39;-H2PO4-(8-oxoG) CUA AAA GU]18.
Finally, it is important to note that mass spectrometry is a powerful method that, through various methodologies, can be adapted to other purposes23,24; thus, choosing the right ionization method as well as other experimental set up is of utmost importance.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.6 mL MCT Graduated Violet</td>
			<td>Fisher Scientific</td>
			<td>05-408-127</td>
			<td></td>
		</tr>
		<tr>
			<td>6&#8217;-Trihydroxyacetophenone monohydrate 98%</td>
			<td>Sigma Aldrich</td>
			<td>480-66-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile 99.9%, HPLC grade</td>
			<td>Fisher Scientific</td>
			<td>75-05-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine triphosphate, 10 mM</td>
			<td>New Englang Bioscience</td>
			<td>P0756S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium citrate, dibasic 98%</td>
			<td>Sigma Aldrich</td>
			<td>3012-65-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Fluoride 98.0%, ACS grade</td>
			<td>Alfa Aesar</td>
			<td>12125-01-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Bruker bacterial test standard</td>
			<td>Bruker Daltonics</td>
			<td>8255343</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial source of Xrn-1</td>
			<td>New England BioLabs</td>
			<td>M0338S</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl pyrocarbonate, 97%</td>
			<td>ACROS Organics</td>
			<td>A0368487</td>
			<td></td>
		</tr>
		<tr>
			<td>Flex analysis software</td>
			<td>Bruker daltonics</td>
			<td></td>
			<td>FlexAnalysis software version 3.4, Bruker Daltonics</td>
		</tr>
		<tr>
			<td>Lambda 365 UV-vis spectrophotometer</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI plate: MSP 96 ground steel target</td>
			<td>Bruker Daltonics</td>
			<td>280799</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass Spectrometer</td>
			<td>Bruker</td>
			<td></td>
			<td>Microflex LRFTOF mass spectrometer (Bruker Daltonics, Billerica, MA)</td>
		</tr>
		<tr>
			<td>Mili-Q IQ 7000</td>
			<td>Milipore Sigma</td>
			<td></td>
			<td>A Mili-Q system was used to purify all water used in this work</td>
		</tr>
		<tr>
			<td>Nanosep Centrifugal Devices with OmegaTM Membrane 10 K, blue (24/pkg)</td>
			<td>Pall Corporation</td>
			<td>OD010C33</td>
			<td>filter media, Omega (modified polyethersulfone) 10 K pore size</td>
		</tr>
		<tr>
			<td>NEBuffer 3</td>
			<td>New England Biolabs</td>
			<td>B7003S</td>
			<td>This is solution B</td>
		</tr>
		<tr>
			<td>Oligo Analyzer tool</td>
			<td>IDT-DNA</td>
			<td></td>
			<td>https://www.idtdna.com/calc/analyzer</td>
		</tr>
		<tr>
			<td>Pipette tips P10</td>
			<td>Fisher Scientific</td>
			<td>02-707-441</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips P200</td>
			<td>Fisher Scientific</td>
			<td>02-707-419</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Away</td>
			<td>Molecular BioProducts</td>
			<td>7005-11</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 Polynucleotide Kinase</td>
			<td>New England BioLabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 Polynucleotide Kinase Reaction Buffer</td>
			<td>New England BioLabs</td>
			<td>B0201S</td>
			<td>This is solution A</td>
		</tr>
		<tr>
			<td>Triflouroacetic Acid</td>
			<td>Alfa Aesar</td>
			<td>76-05-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Xrn-1 exoribonuclease</td>
			<td>Expressed in house</td>
			<td>See ref. 20</td>
			<td></td>
		</tr>
		<tr>
			<td>ZipTip Pipette Tips for Sample preparation</td>
			<td>Millipore</td>
			<td>ZTC 18S 096</td>
			<td>10 &#181;L pipette tips loaded with a C18 standard 0.6 &#181;L bed</td>
		</tr>
	</tbody>
</table>

identification of rna fragments resulting from enzymatic degradation using maldi-tof mass spectrometry

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, and free radicals, are ubiquitous. As a result, RNA undergoes various reactions that include its cleavage, degradation, or modification, leading to biologically relevant species with distinct functions and implications. One example is the oxidation of guanine to 7,8-dihydro-8-oxoguanine (8-oxoG), which may occur in the presence of reactive oxygen species (ROS). Overall, procedures that characterize such products and transformations are largely valuable to the scientific community. To this end, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is a widely used method. The present protocol describes how to characterize RNA fragments formed after enzymatic treatment. The chosen model uses a reaction between RNA and the exoribonuclease Xrn-1, where enzymatic digestion is halted at oxidized sites. Two 20-nucleotide long RNA sequences [5&#39;-CAU GAA ACA A(8-oxoG)G CUA AAA GU] and [5&#39;-CAU GAA ACA A(8-oxoG)(8-oxoG) CUA AAA GU] were obtained via solid-phase synthesis, quantified by UV-vis spectroscopy, and characterized via MALDI-TOF. The obtained strands were then (1) 5&#39;-phosphorylated and characterized via MALDI-TOF; (2) treated with Xrn-1; (3) filtered and desalted; (4) analyzed via MALDI-TOF. This experimental setup led to the unequivocal identification of the fragments associated with the stalling of Xrn-1: [5&#39;-H2PO4-(8-oxoG)G CUA AAA GU], [5&#39;-H2PO4-(8-oxoG)(8-oxoG) CUA AAA GU], and [5&#39;-H2PO4-(8-oxoG) CUA AAA GU]. The described experiments were carried out with 200 picomols of RNA (20 pmol used for MALDI analyses); however, lower amounts may result in detectable peaks with spectrometers using laser sources with more power than the one used in this work. Importantly, the described methodology can be generalized and potentially extended to product identification for other processes involving RNA and DNA, and may aid in the characterization/elucidation of other biochemical pathways.

The oligonucleotides used in this work were synthesized, characterized, and quantified prior to use. The concentration of all oligonucleotides was determined via UV-vis spectroscopy recorded at 90 &#176;C to avoid erroneous readings arising from the potential formation of the secondary structures. Figure 3 displays the spectra of the model oligonucleotides of RNA used in this work, taken at room temperature and after applying heat.
The overall procedure, ...

Watch this Scientific Journal Video about Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry at JoVE.com

Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

MALDI-TOF was used to characterize fragments obtained from the reactivity between oxidized RNA and the exoribonuclease Xrn-1. The present protocol describes a methodology that can be applied to other processes involving RNA and/or DNA.

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, ...

identification-rna-fragments-resulting-from-enzymatic-degradation

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3.2K Views.  University of Colorado, Denver. MALDI-TOF was used to characterize fragments obtained from the reactivity between oxidized RNA and the exoribonuclease Xrn-1. The present protocol describes a methodology that can be applied to other processes involving RNA and/or DNA.

Video: Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important for revealing protease function. The method proposed in this study determines protease specificity by measuring the molecular weight of unique substrates using Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The substrates contain iminobiotin, while the cleaved site consists of amino acids, and the spacer consists of polyethylene glycol. The cleaved substrate will generate a unique molecular weight using a cleaved amino acid. One of the merits of this method is that it may be carried out in one pot using crude samples, and it is also suitable for assessing multiple samples. In this article, we describe a simple experimental method optimized with samples extracted from mouse lung tissue, including tissue extraction, placement of digestive substrates into samples, purification of digestive substrates under different pH conditions, and measurement of the substrates&#39; molecular weight using MALDI-TOF mass spectrometry. In summary, this technique allows for the identification of protease specificity in crude samples derived from tissue extracts using MALDI-TOF mass spectrometry, which may easily be scaled up for multiple sample processing.

Here, we present a protocol to determine protease specificity in crude mouse tissue extracts using MALDI-TOF spectrometry.

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, we developed IDBac&#8212;a low-cost and high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bioinformatics pipeline. IDBac software is designed for non-experts, is freely available, and capable of analyzing a few to thousands of bacterial colonies. Here, we present procedures for the preparation of bacterial colonies for MALDI-TOF MS analysis, MS instrument operation, and data processing and visualization in IDBac. In particular, we instruct users how to cluster bacteria into dendrograms based on protein MS fingerprints and interactively create Metabolite Association Networks (MANs) from specialized metabolite data.

IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite production.

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...