Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

This protocol makes it possible to quantify levels and dynamics of arginine methylation. This modification plays important regulatory roles and could serve as a biomarker for disease. The advantage of this method is that it provides a simple workflow for an almost unlimited number of matrices ranging from cells, media, to tissues and biofluids.

Arginine methylation is elevated in many human cancers and the first inhibitors of arginine method transferases are tested in clinical trials. This method could help with the stratification of patients and risk assessment. Demonstrating the procedure will be Hansjorg Habisch, a research technician from my laboratory.

For liquid samples, add 400 microliters of ice cold methanol to 200 microliters of the sample followed by incubation for 30 minutes at minus 20 degrees Celsius. Centrifuge this sample at 10, 000 G for 30 minutes at four degrees Celsius. Move the supernatant and collect the pellet in 1.5 milliliter tubes.

For solid materials, add 600 microliters of 66%methanol water into pulping tubes containing 30 to 60 milligrams of tissue or cell pellets. Homogenize the soft tissue one time for 20 seconds and the stiff tissue two times for 20 seconds with a five minute interval in between. Immediately place the sample on ice and transfer all lysate to a new 1.5 milliliter tube, followed by incubation.

Then, centrifuge the sample at 10, 000 G for 30 minutes. At four degrees Celsius, select the supernatant in 1.5 milliliter tubes and proceed with protein hydrolysis using this supernatant. Truncate the caps of each tube.

Then add 500 microliters of nine molar hydrochloric acid to each sample. Finally, put the tubes into glass tubes. Close them with caps tightly, ensuring proper sealing, and place these tubes into a glass beaker partly filled with sand.

Hydrolyze the samples for 16 hours at 110 degrees Celsius in a drying chamber. Cool the samples and lyophilize them overnight using a high vacuum centrifuge. On the next day, dissolve completely dried pellets in one milliliter of 0.1 molar hydrochloric acid.

Add 50 microliters of chloroform to each tube and thoroughly dissolve the pellet using a 1, 000 microliter pipette. Transfer the complete volume into a new tube and centrifuge the samples for 10 minutes. Carefully collect the upper phase of the biphasic liquid in a new tube.

Use plastic tubes for manual or glass tubes for robotic solid phase extraction. For manual solid phase extraction, pre-condition the cartridges in every run followed by one minute centrifugation. After that, load the sample onto SPE cartridges and centrifuge them at 600 G for two minutes at room temperature.

Wash the cartridges with one milliliter of water three times, each wash followed by centrifugation for one minute. Then wash five times with one milliliter of 0.1 molar hydrochloric acid, followed by centrifugation for one minute at 800 G at room temperature. Wash the cartridges twice with one milliliter of methanol followed by centrifugation for one minute.

Elute arginine and its derivatives into a single 15 milliliter tube by adding one milliliter of three X replacement solution and centrifuge twice for one minute. For robotic solid phase extraction, place tubes for collection of eluates and the glass tubes containing the upper phase of the biphasic liquid into the respective position of the robot. Use an application or method in the software based on the steps described for manual SPE.

Lyophilize the samples overnight to dry completely to get rid of ammonia and water. Dissolve each sample in 500 microliters of NMR buffer until it becomes homogenous. Then transfer an equal amount of homogenous sample to each NMR tube.

J-RES spectra of yeast protein hydrolysates purified using the SPE protocol are shown here. Different characteristic chemical shifts can discriminate L-arginine and ADMA at 3.25 and 3.02 parts per million. Both substances can be separated and quantified in a cellular matrix.

Based on the number of protons of the specific methyl or methylene group at the respective chemical shift, one H nuclear magnetic resonance spectroscopy allows precise quantification. Using this approach, the one H nuclear magnetic resonance chemical shifts of SDMA and MMA can be separated and quantified by characteristic chemical shifts at 2.76 parts per million SDMA and 2.74 parts per million MMA. When performing this protocol, carefully collect the upper phase of the biphasic liquid containing the water soluble fraction with a pipette and transfer it to new tubes.

Use 1.5 milliliter tubes for manual SPE or class glass for robotic SPE. Avoid spilling over chloroform and its contents. The eluates containing arginine and the methylated arginine species are analyzed using NMR spectroscopy.

This yields absolute amounts of these species. Our technology is currently used in a plethora of basic research projects aiming at dissecting the pathophysiological role of arginine methylation. Furthermore, we are evaluating protein arginine methylation as a biomarker candidate in several human diseases.