Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

This method makes it possible to identify ubiquitylation of EYFP-tagged CENP-A protein. It can also be applied to human CENP-A protein with different tests and other proteins. The main advantage of this is that it can be used to identify EYFP CENP-A K124R ubiquitous sites with crucial biological significance.

It could be extended to investigate the post-translational modifications of a wide range of functional proteins. Begin by preparing protein A beads bound with anti-GFP antibody. Wash 25 microliters of protein A beads per immunoprecipitation reaction with buffer A1 at least three times to remove the ethanol.

Then make a 50%bead solution with buffer A1.Add two microliters of anti-GFP antibody to the beads. Then add buffer A1 to 20 times the net bead volume. Perform end-to-end rotation at four degrees Celsius for four to 18 hours.

The optimal duration for the rotation must be determined empirically based on the efficiency of the immunoprecipitation. After the rotation, centrifuge the beads at 100 times g for one minute and remove the unbound supernatant. Add buffer A1 to make a 50%solution of beads and use 25 microliters of this solution for each immunoprecipitation reaction.

To perform the immunoprecipitation, lyse cells in buffer A1 via sonication and freeze/thaw. Measure protein concentrations and normalize the protein amounts among the different IP samples. Then remove 5%of the sample from each tube to run in SDS page.

Mix the rest of the lysate with 25 microliters of protein A beads bound to anti-GFP antibody and perform end-to-end rotation at four degrees Celsius for four to 18 hours. After lysing cells in buffer A and performing immunoprecipitation as previously described, keep 10%of total immunoprecipitants to confirm EYFP CENP-A ubiquitylation and to precisely determine the position of ubiquitylated EYFP CENP-A. Use the other 90%for mass spectrometry analysis.

Run the mass spectrometry sample in a commercially available 4-12%Bis-Tris protein gel. Then perform Coomassie blue staining and excise the 50-70 kilodalton gel region for mass spectrometry analysis. Dice each gel slice into small pieces and place them in a 0.5 milliliter low protein binding tube.

Wash the gel pieces with 100 microliters of 50%acetonitrile in 25 millimolar ammonium bicarbonate. Vortex them for 10-15 minutes. Then spin them down and discard the supernatant.

After the last wash, dry the gel pieces with a bench top vacuum concentrator for 30 minutes. Add 10 microliters of 10 nanogram per microliter sequencing grade trypsin and let the gel pieces rehydrate for five minutes. Then add 25 millimolar ammonium bicarbonate, just enough to cover the gel pieces and digest them at 37 degrees Celsius overnight.

On the next day, transfer the digested supernatant into a clean 0.65 milliliter siliconized tube and add 50%acetonitrile and 5%formic acid solution. Vortex the sample for 10 minutes. Then spin it down and transfer the supernatant into an extraction tube.

Concentrate the sample again to two microliters. Then add eight microliters of 3%acetonitrile and 2%formic acid solution. Vortex it for 15 minutes and spin it down at 16, 000 times g for 30 minutes.

Perform MS data acquisition with LC-MS/MS using a liquid chromatography system coupled with a mass spectrometry instrument. Inject eight microliters of the sample into a reverse phase liquid chromatography column and separate the peptides with a 2-80%gradient of solvent B in 60 minutes following manuscript directions. Collect mass spectrometry data using the data-dependent acquisition mode.

Open the commercial software to analyze mass spectrometry data on the desktop. To start a new search, click the LC button on the top menu. Then click the add button to upload the original MS raw data files.

Select human protein ID in the paragon method as the database searching method and search the original MS raw data files against the UniProt homo sapiens database. Select trypsin as the digestion enzyme and set the rest of the parameters according to manuscript directions. Enter the results filename and click the save as button on the right of the menu.

Then select a folder for storing the searching results and click the save button. Click the process button to start the search. After the search ends, data with the entered search name will be automatically stored in the selected folder.

To obtain MS/MS spectra of any specific peptide, open the search results in software F.Then click on the protein in the protein list in the top menu and click on the peptide in the middle menu. The MS/MS of this peptide will appear on the bottom of the menu. To export and save the MS/MS spectra, copy the spectra and paste it to a suitable file format.

Gene constructs of EYFP CENP-A wild type or K124R mutant which rescues the loss of endogenous CENP-A were stably expressed when retroviral integration was performed. The expression of endogenous CENP-A was not detected seven days after the induction of Cre recombinase. Both EYFP CENP-A wild type and K124R protein expression was found to be at a similar level to the initial endogenous CENP-A protein.

Both EYFP CENP-A wild type and K124R mutants showed centromere localization at seven days after the disruption of the remaining expression of endogenous CENP-A. The EYFP CENP-A wild type and the K124R mutant showed ubiquitylation and interaction with HJURP unlike the case for flagged tagged or untagged CENP-A wild type and the K124R mutant. Cell viability was assessed by performing the colony outgrowth assay 14 days after the disruption of the remaining endogenous CENP-A allele.

Both EYFP CENP-A wild type and K124R mutants showed a similar number of rescued colonies 14 days after the disruption of the remaining endogenous CENP-A allele. IP mass spectrometry revealed ubiquitylation at lysine 306 in EYFP CENP-A K124R in CENP-A minus F cells. All together, these results suggested that the fusion of large-size protein induces ubiquitylation at a lysine other than K124R in CENP-A which inhibits the original K124R single mutant phenotype.

When attempting this protocol, use the commercially available 4%12%Bis-Tris protein gels to run the mass spectrometry samples. Add adequate trypsin to the gel pieces and rehydrate them until all enzymes have been absorbed. Lower molecular weight tagging or probing technique is actually required to visualize ubiquitylation and investigate spatial temporal protein ubiquitylation signaling at the and entire organism levels.