Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

DNA repair mechanisms are very important for maintaining genome integrity in all living organisms. Uracil-DNA glycosylase is a crucial DNA repair protein in the basic tissue repair pathway. Using MALDI-TOF mass spectrometry, we can directly detect AP product for enzyme activity assay.

Conventional glycosylase assay requires substrate laboring. This method is label-free and directly detect the mass change from a uracil substrate to AP product. Other advantages include high specificity, versatility, scalability, rapidity, and easy to perform.

We have used E.coli uracil-DNA glycosylase as an example. This method can be easily modified for other DNA glycosylase assays. Well-trained laboratory personnel with accurate pipetting and dilution skills are essential.

Freshly prepared reagents and low salt buffers should be used for better signals and lesser background noise. Demonstrating the procedure will be Hui-Lan Chang, a PhD student from my laboratory. For a DNA glycosylase assay using guanine-uracil base-bearing substrate of T1 U+9 duplex, add 70 microliters of water, 10 microliters of 10X uracil-DNA glycosylase or UDG reaction buffer, five microliters of T1 stock, and five microliters of U+9 stock in a 1.5 milliliter sterile microcentrifuge tube.

Close the tube before incubating it in a water bath for 30 minutes at 65 degrees Celsius, followed by 30 minutes at 37 degrees Celsius, and finally on ice for three minutes to ensure proper annealing of the substrate template duplex. Next, in a new 1.5 milliliter tube, add 49 microliters of ice cold UDG reaction buffer and one microliter of diluted UDG, then prepare serial dilutions with the UDG buffer for the desired enzyme concentrations and place the diluted UDG on ice. Next, in another 1.5 milliliter tube, add nine microliters of the prepared substrate mix and prewarm it at 37 degrees Celsius, then add one microliter of the diluted UDG and flick the tube to mix the contents.

Briefly centrifuge the reaction mixture and place the tube immediately for incubation at 37 degrees Celsius. Use the timer to time the reaction. For reaction termination, prepare 0.25 molar hydrochloric acid and 0.23 molar tris base solution.

Use pH meter to confirm the pH when preparing the reagent and testing by pH strip before operating the reaction termination step, then acidify 10 microliters of tris-EDTA with one microliter of 0.25 molar hydrochloric acid and ensure that the pH is around two plus or minus 0.5. Neutralize the solution with one microliter of tris base and ensure that the final pH is around 6.5 plus or minus 0.5. Next, add one microliter of 0.25 molar hydrochloric acid to the reaction mixture to inactivate the enzyme and place the tube on ice for six minutes, then add one microliter of tris base to neutralize the DNA products and avoid AP site breakage by prolonged exposure to acid.

After adding 13 microliters of tris-EDTA to increase the volume of the product mixture for matrix chip transfer, place the tube on ice. Finally, transfer all 25 microliter UDG reaction products from microcentrifuge tubes to a 384-well microtiter plate. Open the door of the nanoliter dispenser and load the 384-well microtiter plate onto the plate holder of the deck, then insert the matrix chip array into the corresponding scout plate position.

Place the loaded scout plate on the processing deck of the nanoliter dispenser and close the door. Touch the run button on the transfer screen and wait for the instrument to start dispensing samples from the microtiter plate to the matrix chip. Next, using the vision tab option, show the image of the chip and the dispense volumes for each spot during dispensing.

Ensure the spotted volume on the chip is in the range of five to 10 nanoliters. Using the appropriate application program, prepare a xlsx file containing the predicted signal M by Z value for importing. Then use the application program to create and define a new UDG assay by right-clicking the import assay group in designer format option and selecting the Excel file from the dropdown list.

Next, right-click the customer project plate button and click on the top of the dropdown option tree to establish a new assay plate. Then in the dialog box, type in a filename. And in the plate type dropdown, select the 384-well plate type.

Press OK and wait for a blank plate to appear on the right of the screen. Next, click the assay option and select the assay from the dropdown list. To assign the selected assay to each sample spot position on the plate, move the cursor to each position of the blank plate, click to highlight the well, and right-click to select add plaques.

Using a desktop or laptop computer, prepare a working list in xlsx format with no header for all the samples on the chip, then click the add new sample project button and select the file from the dropdown list to import the working list. Look for all the test sample codes in the working list on the left of the screen, then click a sample code in the working list and right-click on the corresponding position of the plate to link the tests to each position. Push the in/out button of the mass spectrometer to extend the deck and take out the chip holder.

Insert the sample chip into the chip holder, place the loaded chip holder onto the extended deck and push the in/out button again for the sample chip to enter the instrument. In the mass spectrometer control program, double-click the acquire icon. In the acquire window, click the auto-run tab to start the instrument and acquire mass spectra from the samples on the chip.

For data analysis, run the data analysis program, then browse the database tree and select the chip ID.Click to highlight a target well on the chip and click the spectrum icon to show the mass spectrum. Right-click to choose the customization dialog and crop a specific spectrum range in a new window, then click on the x-axis to type in the upper and lower limits of M by Z and press OK to show the specified range spectrum including the signals of interest. To measure the peak height of the signals M by Z values corresponding to the uracil substrate, AP product, and template, click on the peak and view the peak height in the upper left corner of the screen.

Finally, to save the spectrum for recordkeeping, right-click export and select file type as JPEG in the dropdown list. Click on destination and browse disk to select the storage device in the dropdown list, then type the filename and click export. The model system for a DNA glycosylase assay is shown here.

The 19 nucleotide template DNA remained unchanged after glycosylase hydrolysis. Therefore, the signal could serve as a reference for the quantitation of the AP product. The difference of one nucleotide between the substrate and corresponding template produced a well-separated signal profile for both.

The signal of the AP product was also well-separated from that of the uracil-containing substrate. For MS data analysis, the peak heights were measured. Time course analysis for UDG activity at 0.01, 0.02, 0.05, and 0.1 demonstrated both dose and time dependence.

An example of reaction rate plot with relative MS signal intensities of the product and substrate in nanomolar concentrations is shown here. The UDG reaction rate is presented as nanomoles of the AP site produced per second. The KM and VMAX were calculated from the Lineweaver Burk plot.

The MALDI-TOF mass spectrometry measured unit was proportional to the defined unit from 0.001 units to 0.02 units with an appreciable coefficient of determination. The inhibition of UDG activity by a uracil glycosylase inhibitor is shown here. In the presence of 100 picomoles of the inhibitor, the activity of 0.05 units of UDG was inhibited to an undetectable level and the IC50 was found to be 7.6 picomoles.

Since AP size are labile and can be hydrolyzed by a beta elimination reaction at extreme pH and elevated temperature, assay quenching should be performed on ice for the defined period. Also, use a pH meter to ensure that HCL acidifies the reaction buffer to the desired pH and tris base neutralized the product mixture properly. This method can also be modified to analyze bifunctional DNA glycosylase.

For example, U+2 T3 is a suitable substrate for formamidopyrimidine DNA glycosylase assay. The cleavage products of FPG AP ligase activity can be easily detected by MALDI-TOF mass. This method has the potential to become the reference method for monofunctional glycosylase measurement and can also be used as a tool for glycosylase inhibitor screening for pharmaceutical development.