A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

This protocol is highly significant because it represents the state-of-the-art workflow for the global analysis of protein arginine methylation by mass spectrometry. This is the only method to identify and profile R-methylated protein with a single style resolution which is not achievable with other biochemical techniques. When coupled with the use of PRMT inhibitors, several of which are in clinical trials and anti-cancer drugs, this protocol allows for in-depth analysis of their mechanism of action.

To begin, perform reduction of the thiol group of proteins using a stock solution of DTT dissolved in ultrapure water at a final concentration of 4.5 millimolar and let the reaction go for 30 minutes at 55 degrees Celsius. Perform alkylation of the thiol group of proteins by adding iodoacetamide at a concentration of 10 millimolar and incubating for 15 minutes at room temperature in the dark. To verify the proteolysis efficiency, save an aliquot of protein extract for subsequent analysis on SDS-PAGE Coomassie-stained gel and compare it with a corresponding amount of sample upon digestion.

Dilute the remaining protein extract with four volumes of 20 millimolar HEPES at pH 8.0 to reach a final urea concentration of two molar. Split the sample into two parts, then add sequencing grade modified trypsin to one and LysargiNase protease to the other. Leave the samples overnight at 37 degrees Celsius in a ThermoMixer at 600 rotations per minute to allow for enzymatic digestion.

For each chromatographic gradient, collect all fractions into a deep 96-well plate. Pool the fractions collected before the start of the gradient into one single fraction named pre. Concatenate the 60 fractions from the high pH reversed phase liquid chromatographic gradient by pooling them in a non-contiguous way into 14 final fractions.

To obtain non-contiguous concatenation, pool the high pH reversed phase fractions as described in the text manuscript. Pool the fractions collected after the gradient into a unique fraction named post. Perform sequential immuno-affinity enrichment of the modified peptide separately for the two samples from trypsin and LysargiNase digestions.

Dilute the 10X concentrated immuno-affinity purification or IAP buffer 10 times. Centrifuge the lyophilized peptides at 2, 000 G for five minutes to spin down the peptides to the bottom of the tube. Resuspend the peptides with 250 microliters of diluted IAP buffer per 15 milliliter tube and transfer to a 1.5 milliliter low binding tube.

Use a litmus paper to check that the pH is greater than six. Keep a small aliquot of each fraction as input for the subsequent MS analysis. Split each fraction in two parts to perform the immuno enrichment of asymmetrically dimethylated, or ADMA, and symmetrically dimethylated, or SDMA, peptides in parallel.

Prepare three vials of the selected anti-pan R-methylated antibodies conjugated to protein A agarose beads per 10 milligrams of the initial protein extract. Prepare the correct amount of antibody conjugated to agarose beads by centrifuging each vial at 2, 000 G for 30 seconds and removing the buffer from the beads. Wash the beads three times with one milliliter of 1X PBS by centrifuging them at 2, 000 G for 30 seconds.

After the last wash, resuspend the beads in 40 microliters 1X PBS for each vial, pool them and then finally divide them equally into 16 fractions. Add 250 microliters of 1X IAP buffer to each tube and mix by inverting. Then incubate the tubes on a rotating wheel for two hours at four degrees Celsius.

After the incubation, centrifuge the 1.5 milliliter tubes containing peptides and pan R-methyl antibody conjugated beads at 2, 000 G for 30 seconds to pellet the beads and transfer the flowthrough from each fraction into a clean 1.5 milliliter low binding tube. Add the beads to the flowthrough conjugated to antibodies against R-mono methylation and repeat the resuspension into PBS, incubation with IAP buffer, and centrifugation. During the incubation of the peptide samples with mono-methylization or MMA beads, wash the fractions that were previously immunoprecipitated with anti-ADMA and SDMA with 250 microliters IAP buffer twice and discard the supernatant after each wash.

Then wash with LC-MS grade water three times. Elute the affinity enriched SDMA or ADMA peptides from the agarose beads by adding 50 microliters of 0.15 TFA to each tube. Leave this solution for 10 minutes at room temperature, inverting the tubes every two to three minutes.

Transfer the first elution into clean 1.5 milliliter low binding tubes and repeat the elution with 50 microliters of 0.15 TFA. Pool the two elution fractions into one tube. Repeat this process for the R-mono-methylated peptides that were incubated with the anti-MMA antibody beads.

After mixing the light and heavy labeled cells, proteins were extracted and subjected to digestion by trypsin and LysargiNase. An SDS-PAGE Coomassie-stained gel was used to verify the efficient enzymatic digestion of total proteins in peptides. The efficiency of the purification step performed by C18 Sep-Pak column was evaluated, confirming the absence of peptides in the flowthrough of the C18 column and in the first and second wash as well as their expected presence in the eluent.

Proper MET4 incorporation in the heavy channel and correct heavy or light mixing were evaluated. The chromatogram from the offline high pH reversed phase liquid chromatography fractionation of peptides and the subsequent non-contiguous concatenation of fractions are shown here. Peptides were detected by 250 nanometer UV, while potentially remaining undigested proteins were evaluated by 280 nanometer UV.The full MS spectra of peptides representing true positive methyl peptide annotation were obtained.

The M over Z differences observed between the three peaks are consistent with the presence of enzymatically methylated residue. The full MS spectra of peptides representing false positive methyl peptide annotation were obtained. The M over Z differences observed between the light methylated peptide and its punitive heavy counterpart deviates from the expected value by 0.0312.

The protocol workflow is overall linear, but there are a few steps that needs special attention. For instance, the IPH chromatography separation with non-contiguous fraction concatenation, as well the IP setup. The same protocol can be coupled to either standard SILAC or label-free quantification to profile arginine methylation dynamics in response to a variety of perturbation.

This protocol paves the way to the characterization of the extent and dynamics of protein methylation in several cell types and model systems supporting all aspect of basic and translational research focused on PRMTs.