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

Shawn W. Schowe; Conner J. Langeberg; Erich G. Chapman; Kitty Brown; Marino  J. E. Resendiz

doi:10.3791/63720

JoVE Journal > Biochemistry

Bioquímica

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

Published: April 11, 2022

doi:

10.3791/63720

Shawn W. Schowe¹, Conner J. Langeberg², Erich G. Chapman², Kitty Brown³, Marino J. E. Resendiz¹

¹Department of Chemistry,University of Colorado, Denver, ²Department of Chemistry & Biochemistry,University of Denver, ³Analytical Resources Core Bioanalysis & Omics,Colorado State University

Summary

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.

Abstract

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'-CAU GAA ACA A(8-oxoG)G CUA AAA GU] and [5'-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'-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'-H₂PO₄-(8-oxoG)G CUA AAA GU], [5'-H₂PO₄-(8-oxoG)(8-oxoG) CUA AAA GU], and [5'-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.

Introduction

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'-CAU GAA ACA A(8-oxoG)G CUA AAA GU] and [5'-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'-3' 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'-phosphorylated ends [5'-H₂PO₄-(8-oxoG)G CUA AAA GU], [5'-H₂PO₄-(8-oxoG)(8-oxoG) CUA AAA GU], and [5'-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.

Protocol

RNase-free ultra pure water (Table 1) was used for the present study.

1. Concentration determination of RNA solution

Prepare the RNA sample following the steps below.
1. Use a microcentrifuge tube (0.6 mL) to prepare a solution of RNA by diluting 1 µL of stock solution (obtained via solid-phase synthesis)¹⁹ into 159 µL 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 µL) were used in this work.
2. 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.
3. 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.
Determine the concentration of the prepared RNA solution using UV-vis Spectrophotometer.
1. 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.
2. Turn on the Peltier Temperature Controller spectrophotometer using the switch located to the instrument's right.
3. 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 "Pass" 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.
4. Adjust the scanning parameters by selecting Data Collection. In the new window under Scan Settings change the Start a 350 nm and the End a 215 nm.
5. Activate the Peltier by selecting the + to the left of the Accessory. Click on Multiple Cell Peltier, change the Temperature °C a 25, and click on Peltier On.
6. Navigate to the Sample Info tab and enter the number of samples, names, and cell position as desired.
7. 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.
8. 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 °C.
9. For measurements at higher temperatures, repeat steps 1.2.5-1.2.8 and change the temperature to the desired sample temperature (90 °C in the present case).
10. Click on File > 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).
Perform data acquisition for determining the RNA concentration.
1. Find the file for each spectrum under the Resultados tab. To obtain the absorbance, hover over the line, navigate to 260 nm, and record the displayed absorbances. Alternatively, through the File > Export cascade, export the data as a .txt file for further graphical analysis using other plotting software.
  NOTE: If 0.1 < A ≥ 1, it will be necessary to either dilute or increase the concentration of the RNA sample prepared in step 1.1.1.
2. Use the Beer-Lambert law to calculate the concentration of the solution.
  NOTE: Beer's Law: A = εcl, where:
  A: Absorbance (at 260 nm in this case, from step 1.3.1)
  ε: 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
3. Use these calculations to prepare the desired solution of RNA to be used for subsequent experiments. Solutions with [RNA] = 200 µM were used in the present study.

2. Hydrolysis of oligonucleotides of RNA by Xrn-1

Perform 5' phosphorylation of RNA following the steps below.
1. Use a 0.6 mL microcentrifuge tube to prepare the following solution (50 µL total volume).
  1. Add RNase-free H₂O (33.5 µL); volumes may vary depending on [RNA].
  2. Then, add solution A (5 L, Table 1).
  3. Add an aqueous solution of adenosine triphosphate (ATP, see Table of Materials) (6 L, 10 mM, 60 nmol).
  4. Add RNA aqueous solution (1 µL, 200 µM, 200 pmol).
  5. 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).
2. Incubate the reaction mixture at 37 °C for 45 min by placing it in a water bath.
3. Inactivate the enzyme by placing the reaction tube in a pre-heated heat block (65 °C) and incubating the solution for 10 min.
4. Allow the solution to cool to room temperature (RNA phosphorylation was characterized via MALDI-TOF analysis, see Representative Results) to yield a final 5'-phosphorylated RNA solution (4 µM, 50 µL).
Perform RNA hydrolysis by Xrn-1 following the steps below.
1. Using the solution (50 µL) from step 2.1.4, carry out the following steps.
  1. Add solution B (5 L, Table 1).
  2. Add a solution of Xrn-1 (0.5 L, 1 ng, 30 fmol). Mix the solution by gently pipetting up and down (10x).
  3. Incubate the reaction tube at room temperature for 2 h.
2. 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).
3. Transfer the filtrate into a 0.6 mL centrifuge tube.
4. 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).
5. Combine the filtrate with that obtained from step 2.2.3.
6. Store the tube containing the resulting solution in a freezer (0 °C) or ship for analysis.

3. Desalting of RNA solution, MALDI-TOF plate spotting

Desalt and concentrate the sample using commercially available cation exchange C18 pipette tips (see Table of Materials).
1. Position a 10 L C18 pipette tip (pipette tip loaded with a bed of C18 chromatography media fixed at its end) onto a 10 µL pipette, and then continue with the following:
  1. Wash the C18 tip with 50% aqueous acetonitrile (10 µL) solution two times. Discard the used volumes into a separate waste tube each time.
  2. Equilibrate the C18 tip with an aqueous trifluoroacetic acid solution (0.1% TFA, 10 µL) two times. Discard the used volumes into a separate waste tube each time.
  3. Manually remove the C18 tip from the pipette and secure it onto a 200 µL pipette containing a P200 pipette tip (the C18 tip will now be attached to the P200 pipette tip).
  4. 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.
  5. Detach the C18 tip from the P200 pipette and position it onto a 10 µL pipette.
  6. Wash the C18 tip using an aqueous solution of 0.1% TFA (10 µL) two times. Discard the used volumes into a separate waste tube each time.
  7. Wash the C18 tip with RNase-free water (10 µL) two times. Discard the used volumes into a separate waste tube each time.
Spot onto the MALDI plate following the steps below.
1. Elute the RNA oligonucleotide from the C18 tip (step 3.1.1.7) by immersing the sample into the desired matrix (10 µL, 1:1 solution of C and D, Table 1), followed by dispensing the solution back into the tube. Repeat this process ten times.
2. Pipette the solution from 3.2.1 onto two separate spots on the plate (1 µL 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.
3. 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).
4. Over-lay the [fully dried] calibrant with the desired matrix (1 L). Allow to air dry.
  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)

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.

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).
1. Open "Flexcontrol software" for operation. Press the instrument'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).
2. 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 'Ready', proceed with calibration.
3. To calibrate the instruments using the software, carry out the following.
  1. Select the desired method by clicking on the File > 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).
  2. 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.
  3. 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.
  4. Clear the sum buffer and navigate to the position of the first sample.
  5. Confirm the desired scan range ('Detection' tab). Adjust the bars in the "Mass Range" so that the green region is representative of expected mass(es).
  6. 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).
  7. 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.
4. To obtain spectra, perform the following.
  1. 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).
  2. Click on Add directly below Start.
  3. Repeat this several times until desired intensity/number of shots is reached (reflected on the y-axis of the spectral window)
  4. 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.
  5. 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.
Perform data processing.
NOTE: Step 4.2 describes one way to analyze the data using the flexAnalysis software.
1. Open the desired software. Then, open spectrum (a) by selecting File. From the dropdown menu, select Open.
2. A new window will be displayed. Click on Navegar 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.
3. 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.
4. 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.
5. 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.
6. To add or delete peaks, proceed to the Mass List tab. In the dropdown, click on Edit.
7. 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).
8. 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 > Export > Mass List to Excel.
9. 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.
10. The image generated will reflect the spectral view in the software program. To manipulate the view, perform the following.
  1. Zooming in: Click on the Zoom In button in the toolbar.
  2. Hover over the x-axis and click until the desired range is achieved.
  3. 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).

Representative Results

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 °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, along with the structure of the oxidative lesion that causes Xrn-1 stalling, is illustrated in Figure 4. Oligonucleotide (1) contains one 8-oxoG at position-11 and is where Xrn-1 activity is blocked, resulting in halted hydrolysis of the RNA strand. Importantly, the results depicted in this figure correspond to experiments carried out using solutions containing 200 pmol of the parent strand (1), although only 20 pmol were spotted on the plate. It is important to note that the average mass was used in all of the calculations and that discrepancies varying between 1-3 atomic mass units (amu) were observed. The figure displays two processes, (1) the efficient 5'-phosphorylation of RNA, evidenced by the appearance of only one peak (1') corresponding to the expected product, and (2) the stalling fragment arising from the treatment of the sample mixture with Xrn-1. Control experiments where the parent strand was treated under the same conditions, in the absence of the kinase (PNK), and the ribonuclease (Xrn-1) did not display differences from those shown in Figure 4 (RNA strand 1).

The same results were obtained using a different sequence (3) that contained two oxidative lesions (Figure 5). The corresponding phosphorylation and enzymatic degradation yielded two main products that suggested that the enzyme successively stalls at both sites containing 8-oxoG. As depicted in the mass spectra corresponding to the fragments of interest, a discrepancy upon MALDI-TOF acquisition was observed when taking spectra of the same oligonucleotide on different days. This is illustrated in Figure 4, where two additional atomic units were observed. The results depicted in this figure correspond to experiments carried out with 100 pmol of the parent strand (3). It is important to note that quantitative data may be obtained to assess the overall efficiency of the biochemical process (and complete tracking via MALDI-TOF) by introducing an internal standard on samples before and after enzymatic degradation.

Figure 1: Spotting of RNA solution. (A) Spotting onto the MALDI plate. (B) Illustration of MALDI-plate placement onto the MALDI-spectrometer. Please click here to view a larger version of this figure.

Figure 2: The camera shot illustrating the formation of the sample: matrix crystals spotted onto the plate. Please click here to view a larger version of this figure.

Figure 3: UV-vis spectra of oligonucleotides (1) and (3) recorded at 25 °C and 90 °C, respectively. Please click here to view a larger version of this figure.

Figure 4: Experimental process for oligonucleotide preparation and mass spectra for each step. (A) Overall steps using oligonucleotide (1) are illustrated. The sequence of events corresponds to (B) 5'-phosphorylation in the presence of polynucleotide kinase (PNK), followed by (C) enzymatic hydrolysis in the presence of Xrn-1. The MALDI-TOF spectra obtained before and after both biochemical processes are displayed. Please click here to view a larger version of this figure.

Figure 5: MALDI-TOF spectra, using oligonucleotide (3), obtained before (left)/after (right) treatment with Xrn-1. Please click here to view a larger version of this figure.

Solution	Composition
A	Tris-HCl (700 mM), DTT (50 mM), pH 7.6, T4 polynucleotide kinase buffer
B	NaCl (1 M), Tris-HCl (0.5 M), MgCl₂ (0.1 M), DTT (10 mM), pH 7.9
C	2,4,6-Tihydroxyacetophenone monohydrate (THAP, 25 mM), ammonium citrate (10 mM)
D	Ammonium fluoride (300 mM in 50 % aq. Acetonitrile)
RNase-free H₂O	Ultra-pure water was treated with diethyl pyrocarbonate (DEPC, 1 mL per liter of water), incubated at 37 °C (12 h), and autoclaved (1 h)

Table 1: Solution compositions used in the study.

Video 1: Demonstration for spotting onto the MALDI plate. Please click here to download this Video.

Video 2: Demonstration outlining the data analyses. Please click here to download this Video.

Supplementary File 1: Step-by-step illustration for handling UV-vis software (step 1.2), MS data acquisition (step 4.1), and MS data processing (step 4.2). Please click here to download this File.

Discussion

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 to spotting and acquiring; however, this time delay did not seem to affect the experimental outcome, although degradation of the RNA (leading to decreased signal intensities) cannot be ruled out.

The limitation of the described experiments, in terms of RNA concentration, was that the experiments were carried out with [RNA] higher than what can be obtained in vivo, for a single RNA sequence²³^,²⁴. We found that when experiments were carried out with 20 pmol of RNA (2 pmol spotted on the plate), or lower amounts, the expected peaks were not observed. The use of spectrometers with improved laser power may likely lead to enhanced signal and lower detection limits.

It is expected that the step where the enzymes are filtered (using a 10 kDa filtering device) to separate the enzymes can be avoided. Experiments carried out without this step led to similar results. However, the filtration step is recommended if samples are to be shipped or stored for a while.

The provided steps may be amenable to characterizing and understanding several biochemical processes. Examples may include the characterization of processes involving damaged DNA²⁵, peptides²⁶^,²⁷, or RNA conjugates²⁸, among many others. In general, we speculate that the process illustrated herein may be applicable to various experimental setups involving nucleic acids, proteins, biomaterials, and/or other biopolymers. Furthermore, the development of enhanced spectrometers will lead to enhanced results, and important discoveries, in areas involving the aforementioned biomolecules.

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

Referencias

Tanaka, K., et al. Protein and polymer analyses up to m/z 100 000 by laser ionization time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry. 2 (8), 151-153 (1988).
Karas, M., Hillenkamp, F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Analytical Chemistry. 60 (20), 2299-2301 (1988).
Zenobi, R. Chemistry nobel prize 2002 goes to analytical chemistry. Chimia. 57, 73 (2003).
Aristri, M. A., et al. Bio-based polyurethane resins derived from tannin: Source, synthesis, characterization, and application. Forests. 12 (11), 1516 (2021).
Pedrazzani, C., et al. 5-n-Alkylresorcinol profiles in different cultivars of einkorn, emmer spelt, common wheat, and tritordeum. Journal of Agricultural and Food Chemistry. 69 (47), 14092-14102 (2021).
Unger, M. S., Blank, M., Enzlein, T., Hopf, C. Label-free cell assays to determine compound uptake or drug action using MALDI-TOF mass spectrometry. Nature Protocols. 16 (12), 5533-5558 (2021).
Croxatto, A., Prod’hom, G., Greub, G. Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology. FEMS Microbiology Reviews. 36 (2), 380-407 (2012).
Kaufmann, R. Marrix-assisted laser desorption ionization (MALDI) mass spectrometry: a novel analytical tool in molecular biology and biotechnology. Journal of Biotechnology. 41 (2-3), 155-175 (1995).
Kiggins, C., Skinner, A., Resendiz, M. J. E. 8-Oxo-7,8-dihydroguanosine inhibits or changes the selectivity of the theophylline aptamer. ChemBioChem. 21 (9), 1347-1355 (2020).
Chapman, E. G., DeRose, V. J. Enzymatic processing of platinated RNAs. Journal of the American Chemical Society. 132 (6), 1946-1952 (2010).
Resendiz, M. J. E., Pottiboyina, V., Sevilla, M. D., Greengerg, M. M. Direct strand scission in double stranded RNA via a C5-pyrimidine radical. Journal of the American Chemical Society. 134 (8), 3917-3924 (2012).
Joyner, J. C., Keuper, K. D., Cowan, J. A. Analysis of RNA cleavage by MALDI-TOF mass spectrometry. Nucleic Acids Research. 41 (1), 2 (2013).
Ghodke, P. P., Guengerich, P. DNA polymerases η and κ bypass N2-guanine-O6-alkylguanine DNA alkyltransferase cross-linked DNA peptides. Journal of Biological Chemistry. 297 (4), 101124 (2021).
JoVE. JoVE Science Education Database. Biochemistry. MALDI-TOF Mass Spectrometry. Journal of Visualized Experiments. , (2021).
Schrötner, P., Gunzer, F., Schüppel, J., Rudolph, W. W. Identification of rare bacterial pathogens by 16S rRNA gene sequencing and MALDI-TOF MS. Journal of Visualized Experiments: JoVE. (113), e53176 (2016).
Su, K. -. Y., et al. Proofreading and DNA repair assay using single nucleotide extension and MALDI-TOF mass spectrometry analysis. Journal of Visualized Experiments: JoVE. (136), e57862 (2018).
Fagerquist, C. K., Rojas, E. Identification of antibacterial immunity proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down proteomic analysis. Journal of Visualized Experiments: JoVE. (171), e62577 (2021).
Phillips, C. N., et al. Processing of RNA containing 8-Oxo-7,8-dihydroguanosine (8-oxoG) by the exoribonuclease Xrn-1. Frontiers in Molecular Biosciences. 8, 780315 (2021).
Francis, A. J., Resendiz, M. J. E. Protocol for the solid-phase synthesis of oligomers of RNA containing a 2′-O-thiophenylmethyl modification and characterization via circular dichroism. Journal of Visualized Experiments: JoVE. (125), e56189 (2017).
Langeberg, C. J., et al. Biochemical characterization of yeast Xrn1. Bioquímica. 59 (15), 1493-1507 (2020).
Stevens, A. Purification and characterization of a Saccharomyces cerevisiae exoribonuclease which yields 5′-mononucleotides by a 5′ leads to 3′ mode of hydrolysis. Journal of Biological Chemistry. 255 (7), 3080-3085 (1980).
Yan, L. L., Simms, C. L., McLoughlin, F., Vierstra, R. D., Zaher, H. S. Oxidation and alkylation stresses activate ribosome-quality control. Nature Communications. 10 (1), 5611 (2019).
Fasnacht, M., et al. Dynamic 23S rRNA modification ho5C2501 benefits Escherichia coli under oxidative stress. Nucleic Acids Research. 50 (1), 473-489 (2022).
Estevez, M., Valesyan, S., Jora, M., Limbach, P. A., Addepalli, B. Oxidative damage to RNA is altered by the presence of interacting proteins or modified nucleosides. Frontiers in Molecular Biosciences. 8, 697149 (2021).
Tomar, R., et al. DNA sequence modulates the efficiency of NEIL1-catalyzed excision of the aflatoxin B1-induced formamidopyrimidine guanine adduct. Journal of the American Chemical Society. 34 (3), 901-911 (2021).
Gaffney, A., et al. HIV-1 env-dependent cell killing by bifunctional small-molecule/peptide conjugates. ACS Chemical Biology. 16 (1), 193-204 (2021).
Sikorski, E. L., et al. Selective display of a chemoattractant agonist on cancer cells activates the formyl peptide receptor 1 on immune cells. ChemBioChem. , 202100521 (2022).
Kubo, T., Nishimura, Y., Sato, Y., Yanagihara, K., Seyama, T. Sixteen different types of lipid-conjugated siRNAs containing saturated and unsaturated fatty Acids and exhibiting enhanced RNAi potency. ACS Chemical Biology. 16 (1), 150-164 (2021).

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Schowe, S. W., Langeberg, C. J., Chapman, E. G., Brown, K., Resendiz, M. J. E. Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry. J. Vis. Exp. (182), e63720, doi:10.3791/63720 (2022).