Ubiquitin Chain Analysis by Parallel Reaction Monitoring

As different ubiquitin chain topologies have a broad range of biological effects, understanding changes in their abundance is relevant to a wide variety of biological states. Applying parallel reaction monitoring to ubiquitin chain analysis allows the specific identification and quantification of the ubiquitin chain topologies. Unlike typical PRM experiments, analysis of ubiquitin chain topology is restricted to specific modified peptides, many of which are not ideal for MS analysis.

Visual demonstration of this technique highlights the adjustments and the expected results observed for these non-ideal peptides. Begin by preparing an ammonium bicarbonate solution, n-ethylmaleimide solution, and ubiquitin stabilization buffer as described in the text manuscript. Resuspend the biological sample in the ubiquitin stabilization buffer and lyse the cells with an appropriate method.

After cell lysis, centrifuge the sample at 18, 000 times G for 10 minutes at four degrees Celsius and transfer the supernatant to a fresh low protein binding microcentrifuge tube. If necessary, store the samples at negative 20 degrees Celsius before proceeding. To thaw the samples, mix them rapidly with a ThermoMixer and do not use temperatures above 50 degrees Celsius.

Once thawed, centrifuge the samples at 18, 000 times G for 10 minutes at four degrees Celsius and transfer 20 micrograms to a fresh low bind microcentrifuge tube, then adjust the volume to 50 microliters with ubiquitin stabilization buffer. Prepare a negative control with a normalized volume of ubiquitin stabilization buffer and a positive control with the buffer and commercially available chain types. Prepare a solution of 50 millimolar ammonium bicarbonate in water and adjust the pH to eight with one molar sodium hydroxide.

Pellet an E.coli culture by centrifugation at 5, 000 times G for five minutes. Wash the cells twice with PBS and resuspend them in ubiquitin stabilization buffer at five micrograms per milliliter. Add one microgram of E.coli lysate to each sample and control.

Then prepare 500 millimolar TCEP in MS Grade water and add it to each sample to a final concentration of 50 millimolar. Vortex the samples briefly and incubate them for 30 minutes at room temperature. Next, prepare a solution of 500 millimolar chloroacetaldehyde and ammonium bicarbonate and add it to the samples and controls to a final concentration of 55 millimolar.

Vortex the samples briefly and incubate them in the dark for 20 minutes. Add Endopeptidase Lys-C to the samples and incubate them at 37 degrees Celsius for three hours. After the incubation, dilute the samples with 200 microliters of 50 millimolar ammonium bicarbonate, add trypsin, and incubate them for another 12 hours.

On the next day, add 10%formic acid solution to each sample and control at a 1:10 volume-to-volume ratio. Verify that the pH is less than three. Then add 0.5 microliters of heavy peptide standard to each sample.

After standard C18 sample cleanup, proceed with mass spectrometry analysis of the samples with a PRM-based methodology. To export the isolation list, select isolation list from the export menu, select the instrument type and set the method to standard. Click OK which will open a prompt to save a CSV file that can be used to create a PRM method.

Next, import the scheduling run by selecting File, Import, and Results. Choose several files to import simultaneously and click OK, then select the files and wait for the import to complete. When ready, review the identifications by clicking on the mass for each heavy peptide entry.

Correct recognition of the peak is often determined automatically, but manual curation may be required. Retention times selected for the heavy variants are also applied to the light versions, creating a schedule for the PRM. To modify the scheduling window, select the peak and click on Settings and Peptide Settings.

Then change the time window in the Prediction tab. The schedule inclusion list can then be exported using File, Export, and Isolation List. Choose the appropriate instrument type, set method type to scheduled, and click OK.To analyze the data, import all samples and perform curation, removing transitions with interference or poor signal-to-noise ratios, then export data by either right-clicking on relevant graphs and selecting Copy Data or clicking on File, Export, and Report.

Treatment with the proteasome inhibitor MG-132 prevents the degradation of ubiquitin conjugated proteins, which results in an increase of the K48 chain in the mouse melanoma cell line B16, as well as the two human cell lines, A549 and HeLa. Parallel reaction monitoring or PRM performs a full product ion scan after selection of the precursor ion, which means that product ions should be curated post run. The chromatogram for K48 is shown before and after curation.

Product ions that have a signal with an inconsistent elution profile and low-intensity were removed. The transition selected during curation should be consistent between experiments, but may differ depending on chromatographic conditions, analysis settings, and the biological background of the sample. Shown here are typical product ion chromatograms for each of the identified ubiquitin chain topologies in this experiment.

When designing a PRM experiment, collision energy can be optimized to improve the signal. When collision energies from 14 to 28 were applied, it was found that a higher collision energy of 26 was optimal for K63, while a lower energy of 18 was ideal for M1.Favorable collision energies were found for the topology characteristic peptides in this experiment and a selection of unmodified ubiquitin fragments, but they may need to be optimized based on the mass spectrometer and fragmentation method used. While attempting this procedure, it is important to remember that for ubiquitin chain analysis, a balance of conditions must be created to ensure that ubiquitin chain linkages are maintained whilst promoting their digestion into peptides.

This method is vital for the understanding of ubiquitin signaling as it provides an accurate quantification of the ubiquitin chain topology. It could be combined with a pull-down or an organelle fractionation to address which ubiquitin chain topology is associated with the targeted protein sample.