Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

This protocol describes the novel technique for simultaneous enrichment of multiple PTMs with antibodies followed by data independent acquisition mass spectrometry analysis to gain biological insight into site localization and cross-talk. This is a time and cost effective technique which enables the simultaneous identification and quantification of peptides with slicing containing acetylation and succinylation PTMs with only small amounts of sample input. Tracking post-translational modifications is an important step in characterizing and combating different disease states.

We're already aware that certain cancers and diabetes are highly regulated through these protein modifications. This method can theoretically apply to any PTMs with antibodies for enrichment such as ubiquitination, phosphorylation and others. It can also apply to other tissue types or cell culture models.

The key to obtaining valuable data using this technique is reproducibility. This can be achieved by ensuring all steps are performed very carefully particularly the steps involving the antibody beads. To begin, obtain cartridges containing C18 resin that combined up to 10 milligrams of protein.

Fit these cartridges into a vacuum apparatus. Add 800 microliters of 80%acetonitrile and 0.2%formic acid to the cartridges with 19.8%water and start vacuum suction to pull the liquid through. Repeat this step one time avoiding drying of the cartridges completely.

Equilibrate the cartridges by adding 800 microliters of 0.2%formic acid in water with vacuum suction. Repeat this twice. Load one milligram peptides in about 1, 000 microliter solution onto the cartridges with vacuum suction.

Wash the peptides twice with 800 microliters of 0.2%formic acid in water under vacuum suction. Then arrange 1.5 milliliter microcentrifuge tubes beneath each cartridge to collect the peptide. Under vacuum suction, elute peptides from cartridges first with 800 microliters of 80%acetonitrile and 0.2%formic acid in 19.8%water then with 400 microliters of the same solution.

Dry the desalted peptide samples completely in a vacuum concentrator for two to three hours. Now, resuspend the dried peptides in 1.4 milliliters of cold 1X immunoaffinity purification buffer. Vortex to mix and ensure a pH of approximately 7.

Centrifuge the samples at 10, 000 times g for 10 minutes at four degrees Celsius. A small pellet appears. Set the peptides aside on ice while preparing antibody beads.

Add one milliliter of cold 1X PBS to a tube containing 100 microliters of K-acetyl antibody bead slurry and a tube containing 100 microliters of K-succinyl antibody bead slurry and mix by pipetting. Transfer the entire solution to new 1.5 milliliter tubes and spin in a miniature centrifuge for 30 seconds at room temperature. Aspirate the PBS taking care to avoid aspirating off any beads.

Repeat the wash three additional times. Next, resuspend the beads in about 440 microliters of PBS. Pipette the beads several times to mix well.

With 200 microliter precut tips, transfer 100 microliters of each acetyllysine antibody beads and succinyllysine antibody beads into a new tube. Spin down for 30 seconds in a miniature centrifuge and aspirate off the media with a gel loader tip. The beads now turn white.

Pipette the resuspended peptide directly onto the washed beads and incubate the peptides with beads at four degrees Celsius overnight with agitation. In the morning, spin the peptide samples at 2, 000 times g for 30 seconds at four degrees Celsius. Remove the supernatant containing unbound peptides and save for future experiments if desired.

Add one milliliter of cold 1X immunoaffinity purification buffer to the beads to wash and mix by inverting five times. Spin at 2, 000 times g for 30 seconds at four degrees Celsius. Aspirate off the immunoaffinity purification solution and repeat the immunoaffinity purification wash step one more time.

Then add one milliliter of cold HPLC water to the beads and mix by inverting five times. Spin at 2, 000 times g for 30 seconds at four degrees Celsius. Aspirate off the water and repeat the water wash step two additional times.

Spin an additional time at 2, 000 times g for 30 seconds at four degrees Celsius to collect any remaining water in the bottom of the tube. With flat tipped gel loading tips, aspirate off any extra water that have collected. Be careful to avoid aspirating the beads.

Add 55 microliters of 0.15%trifluoroacetic acid in water to the beads. Incubate the beads for 10 minutes at room temperature while occasionally tapping the bottom of the tube to mix. Spin the beads at room temperature for 30 seconds in a miniature centrifuge.

Use a flat tipped gel loading tip to transfer the eluted peptides into a new tube. Add 45 microliters of 0.15%trifluoroacetic acid in water to the beads. Incubate for 10 minutes at room temperature while tapping the bottom of the tube occasionally to mix.

Spin the beads at room temperature for 30 seconds in the miniature centrifuge. Remove the second elution using a flat tipped gel loading tip and combine it with the first elution. Spin the combined eluted peptides at 12, 000 times g for five minutes at room temperature to pellet any beads that carry over.

Transfer the peptide sample solution into a new 500 microliter tube. Build the loading method to load the enriched peptide mixtures onto a C18 pre-column chip and desalt the peptides again by washing with mobile phase A at two microliters per minute for 10 minutes. Build the chromatography method such that peptides are transferred to an analytical column and elute at a flow rate of 300 nanoliters per minute with a two to three-hour gradient using mobile phases A and B.Specifically, use a linear gradient from 5%mobile phase B to 35%mobile phase B over 80 minutes.

Subsequently, ramp the mobile phase B to 80%over five minutes then hold at 80%B for eight minutes before returning to 5%B for a 25-minute re-equilibration. Next, build an MS instrument method for data-dependent acquisition. For experiment one, set up MS1 precursor ion scan from M/Z 400 to 1, 500.

Set the duration to 120 minutes. Create an IDA experiment and click OK.Under the switch criteria tab, set the intensity threshold to trigger MS/MS scans for ions of charged states between two to five to 200 counts per second. Set the dynamic exclusion of precursor ions to 60 seconds.

For experiment two, set MS/MS product ion scan with an MS2 scan range from M/Z 100 to 1, 500. Click edit parameters and set the collision energy spread to five and select the high sensitivity product ion scan mode. Finally, place the sample into the autosampler.

Submit the sample queue and start LC-MS/MS acquisition methods building an MS instrument method for data-independent acquisition and defining two instrument scan experiments. Perform MS1 precursor ion scans from 400 to 1, 250 mass to charge and set the duration to 120 minutes. Set the collision energy spread to 10 and accumulation time to 45 milliseconds then select the high sensitivity product ion scan mode.

Next, import a text file containing the 64 variable window DIA SWATH acquisition scheme to populate SWATH acquisition windows into the method. In this acquisition scheme, variable windows ranging from five to 90 mass to charge and width are passed in incremental steps over the full mass range of 400 to 1, 250 mass to charge with a total of 64 SWATH segments each with a 45 millisecond accumulation time. Adjust the MS1 accumulation time to 0.25 seconds.

Together, the MS1 scan and 64 MS2 scans should now yield a total cycle time of 3.2 seconds. In this study, experimental results documented the possibility of detecting and assessing PTM cross-talk. This table shows how the timeline of the workflow and the amounts of sample and protein required.

The one-pot method can be performed in half as much time and with half the number of samples as these alternative methods. The median coefficient of variation for the modified peptide areas was lower in the one-pot method than in the single PTM and serial PTM enrichment. While comparing the one-pot PTM and single PTM enrichment methods, no noteworthy differences were apparent between the correlations of site-level quantification for the two modifications.

This figure displays data from a successful enrichment and illustrates an example for a peptide containing multiple and different acyl modifications visualizing PTM cross-talk. A peptide was acetylated on one lysine residue and succilynated on the other while here the same peptide was succilynated at both lysines. This demonstrates that the same lysine residue can be modified with both acylation groups and there is a possibility of cross-talk occurring at that site.

It is essential to make sure that each sample contains the same amount of beads and make sure that none of the beads are aspirated accidentally when washing peptide bond beads. Mass spectrometry based profiling and data analysis in software tools such as Spectronaut are performed following this procedure to identify, quantify and perform site localization of PTMs. This data-independent acquisition workflow in combination with simultaneous PTM enrichment will open avenues to more efficiently assess PTM cross-talk and provide better insights into the relevance of PTM signaling.