Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The post translational modification of proteins by the small protein ubiquitin is involved in many events in the cell. This study presents an original addition to the toolbox for protein ubiquitination analysis. We use enrichment techniques and mass spectrometry to uncover the deep ubiquitinome.

We have made several improvements to the analysis of diGly peptides that originate from ubiquitinated proteins. These include crude peptide fractionation prior to enrichment, and the application of more advanced peptide fragmentation settings in the Orbitrap. Altogether this results in a larger coverage of the ubiquitinome.

Several aspects of the protocol are difficult to describe in words. Visualization of some of the key steps is essential to be able to repeat these analyses by different operator in a different lab. The procedure will be demonstrated by Karel Bezstarosti, who is a senior technician in my lab.

Begin by preparing cultured cells or mouse brain tissue for the experiment. If working with cells, lyse the cell pellet from one 150 centimeter squared culture plate in two milliliters of ice cold, 50 millimolar Tris HCL with 0.5%sodium deoxycholate. Boil the lysate at 95 degrees Celsius for five minutes.

Then sonicate it at four degrees Celsius for 10 minutes. If using in vivo mouse brain tissue, lyse it in an ice cold buffer containing 100 millimolar Tris HCL, 12 millimolar sodium deoxycholate and 12 millimolar sodium N-lauroylsarcosinate. Sonicate the lysate for 10 minutes at four degrees Celsius.

Then boil it for five minutes at 95 degrees Celsius. Next, quantitate the total protein amount using a calorimetric absorbance BCA protein assay kit, which should be at least several milligrams for successful diGly peptide immunoprecipitation. For SILAC experiments, mix the light and heavy labeled proteins in a one to one ratio based on the total protein amount.

Reduce all proteins with five millimolar DTT for 30 minutes at 50 degrees Celsius. And subsequently alkylate them with 10 millimolar iodoacetamide for 15 minutes in the dark. Then perform protein digestion with Lys-C for four hours.

And trypsin digestion overnight at 30 degrees Celsius, or at room temperature. On the next day, divide the sample into two two milliliter Eppendorf tubes and add TFA to the digested sample to a final concentration of 0.5%Centrifuge it at 10, 000 times G for 10 minutes to precipitate and remove all detergent. Collect the peptide containing supernatant for subsequent fractionation.

Use high pH reverse phase C18 chromatography to fractionate the tryptic peptides. Prepare an empty six milliliter column cartridge filled with 0.5 grams of stationary phase material for about 10 milligrams of protein digest. Load the peptides onto the prepared column and wash it with approximately 10 volumes of 0.1%TFA, followed by 10 volumes of water.

Elute the peptides into three fractions using 10 column volumes of 10 millimolar ammonium formate with seven, 13.5 and 50%acetonitrile, respectively. Then, litholyse all fractions. One batch of ubiquitin remnant motif antibodies conjugated to protein A agarose bead slurry is split into six equal fractions.

Dissolve the three peptide fractions according to the manuscript directions and spin down the debris. Add the supernatants of the three fractions to the bead slurry while keeping the other three fractions on ice. And incubate them for two hours at four degrees Celsius on a rotator unit.

Then, spin down the beads. Transfer the supernatant to a fresh batch of beads. And repeat the incubation with the remaining three bead slurry fractions.

Store the supernatants for subsequent global proteome analysis. And transfer the beads to 200 microliter pipette tips equipped with a GFF filter plug. Put the tips into 1.5 milliliter tubes equipped with a centrifuge tip adaptor and wash the beads three times with 200 microliters of ice cold IAP buffer, followed by three washes with ice cold purified water.

Spin down the columns at 200 times G for two minutes between washes, making sure not to let the column run dry. After the final wash, elute the peptides with two cycles at 50 microliters of 0.15%TFA. Desalt the peptides with a C18 stage tip and dry them with vacuum centrifugation.

Perform LC-MS/MS experiments on a sensitive mass spectrometer coupled to a nanoflow LC system. The column is set up according to manuscript directions and kept at 50 degrees Celsius. Operate the mass spectrometer in data dependent acquisition mode.

Collect the MS1 mass spectra at high resolution with an automated gain controlled target setting of 4E5 and a maximum injection time of 50 milliseconds. Perform the mass spectrometry analysis in most intense first mode, using the top speed method with a total cycle time of three seconds. Then perform a second round of DDA MS analysis in least intense first mode, which will ensure optimal detection of low abundance peptides.

Filter the precursor ions according to their charge states and monoisotopic peak assignment. And exclude previously interrogated precursors dynamically for 60 seconds. Isolate peptide precursors with a quadrupole mass filter set to a width of 1.6 Thomson.

Then collect MS2 spectra in the ion trap at an automated gain control of 7E3 with a maximum injection time of 50 milliseconds and HCD collision energy of 30%Analyze the mass spectrometry raw files using an appropriate search engine, such as the freely available MaxQuant software suite based on the Andromeda search engine. Open MaxQuant and select the appropriate raw data files. Set the number of processors and click on the group specific parameters tab.

Select digestion, and allow for three missed cleavages. Then select modifications and add diGly to the variable modifications. Select the global parameters tab and add the correct protein sequence database.

Leave the other settings as default and press start to perform the database search. For quantitative analysis of SILAC experiment files, set the multiplicity to two, and select the heavy amino acid labels. When the search is finished, import the text files into Perseus.

This protocol was used to identify ubiquitination sites in proteins from cultured cells and in vivo material by detecting diGly peptides with nanoflow LC-MS/MS. Several improvements were made to the existing protocol which resulted in higher numbers of detected diGly peptides. A crude fractionation into three fractions was performed prior to immunoprecipitation.

One of the fractions contains ubiquitin's own K48 modified tryptic diGly peptide, which is characterized by a broad peak in the LC chromatogram. Furthermore, the peptide fragmentation regime was adjusted to combine the LC-MS runs with the highest first and lowest first fragmentation regimes in the data analysis procedure, which produced more than 4, 000 additional unique diGly peptides. More than 23, 000 diGly peptides can be routinely identified from a single sample of HeLa cells treated with a proteasome inhibitor.

Of all diGly peptides identified over three biological replicate screens, more than 9, 000 were present in all three, while more than 17, 000 were present in at least two out of three replicates. The number of peptide identifications is highly contingent on the amount of input material. An approximate number of identified diGly peptides can be expected depending on the starting material, but these numbers are only estimations and will also depend on the type of mass spectrometer used.

When carrying out this protocol, prior experience with mass spectrometry based proteomics methods and handling and analyzing minute sample amounts would certainly be advantageous. The MaxQuant software could be replaced by any other database searching algorithm. Also a different type of mass spectrometer may be used.

The results in terms of peptide identifications may slightly differ, but this is typical for any mass spectrometry based proteomics assay. Ubiquitin is important in progress of mediated protein degradation, but also plays a role in many other processes in the cell. Deeper knowledge of ubiquitin as a post translational modification is crucial to better understand its function.