The overall goal of this procedure is to identify protein interaction partners for a given protein of interest. This is accomplished by first growing the cells in cell culture media containing arginine and lysine labeled with different stable isotopes of carbon and nitrogen. The second step is to transfect, plasmids and coating a protein of interest or a control plasmid into the labeled cells.
Next, the cells are lysed and samples are immuno precipitated from the lysates. Then equal volumes of control and sample immuno precipitates are combined and submitted for lc MS MS analysis. The final step is to analyze the mass spectrometry data to identify proteins interacting with the protein of interest.
Ultimately, stable isotope labeling of amino acids in cell culture or silac amino precipitations are capable of identifying large numbers of both direct and indirect interactions with a protein of interest from tissue culture cells. The main advantage of this technique over existing methods, such as tap tagging, is that it removes the need for the investigator to send away individual bands from mass spectrometry. This removes an important source of bias whilst increasing sensitivity.
Though this method can be used to provide insight into cellular protein, protein interactions can also be applied to other systems such as studying pathogen host interactions. To begin scrape SLAC labeled HEK 2 93 T cells, expressing the GFP tagged protein of interest or GFP control from the cell culture dish into ice cold PBS then centrifuge the cell suspension at 220 times G for five minutes at four degrees Celsius, wash the cells three additional times in 10 milliliters of ice cold PBS resuspend the cell pellet in 200 microliters of cell lysis buffer containing freshly added protease inhibitor cocktail three at one X concentration and add five microliters of RNAs cocktail per milliliter of lysis buffer, incubate on ice for five minutes. Then centrifuge the cell lysate at 13, 000 times G for 10 minutes at four degrees Celsius and retain the supernatant as the soluble cell lysate.
Measure the protein concentration of the cell lysate using A BCA assay. Then normalize the protein concentration in each sample lysate in a final volume of 500 microliters using the lysis buffer containing protease inhibitors. Add 500 microliters of dilution buffer containing one x protease inhibitor cocktail three to each of the normalized cell lysates to adjust the total volume to one milliliter.
Keep lysates on ice while preparing anti GFP beads and retain a 50 microliter aliquot of each sample to be used for sample input. Vortex the bead slurry to re suspend the beads. Then use a 200 microliter pipette tip with the end cutoff to transfer 25 microliters of beads per sample to a fresh tube.
For each 25 microliters of bead slurry, add 20 volumes of dilution buffer to wash, centrifuge the beads at 2, 700 times G for five minutes. Wash the beads to additional times with 20 volumes of dilution buffer. Next, add 100 microliters of dilution buffer per 25 microliters of bead slurry.
Then use a 200 microliter pipette tip with the end cutoff to transfer 85 microliters of the resuspended bead slurry to each of the SLAC labeled sample Lysates. Incubate the samples with beads on a rotator at four degrees Celsius for two hours after the samples have incubated with the beads, centrifuge the samples at 2, 700 times G for five minutes at four degrees Celsius. Retain 50 microliters of the supernatant as the unbound sample and discard the remainder of the supernatant.
Resuspend the beads in each tube in one milliliter of dilution buffer, and then centrifuge at 2, 700 times G for five minutes at four degrees Celsius. Repeat this wash step twice to elute the protein from the beads. Add 50 microliters of two XSDS loading buffer to each sample and heat at 95 degrees Celsius for 10 minutes.
Once the heating is finished, centrifuge each sample at 2, 700 times G for five minutes at four degrees Celsius. Transfer the supernatant to pre lubricated tubes and store at negative 80 degrees Celsius until ready to submit for MS analysis. Then mix equal volumes of control and sample amino precipitates and submit the combined sample to a mass spectrometry facility for lc ms MS analysis.
Prior to analyzing the mass spectrometry data, copy the raw data into a new spreadsheet. Then in this spreadsheet, remove all the columns except for the columns containing the accession number, number of unique peptides ratios, comparing samples, ratio variability, and the protein description. Use the Excel sort function to order the data by the number of peptides and remove the entries for proteins lacking more than one peptide.
Then sort by ratio and remove proteins that lack SLAC ratios. Next, convert slac ratios to log two values. Using the formula equals log parentheses, SLAC ratio two and parentheses where SLAC ratio is substituted for the cell identifier.
Then create a new column in the Excel file and calculate Log two SLAC ratios for all the sample mock columns for conversion of a mock sample ratio to a sample mock ratio. Use the formula equals one divided by ratio. Open GraphPad prism.
Select new data table and graph Select column from the list on the left hand side of the window and select the enter import data. Enter replica values stacked into columns options. Press create.
Then select and copy a given log two sample mock SLAC ratio column from the Excel spreadsheet into the new prism file. Next, click the dropdown insert menu and select new analysis. Under column analysis, select frequency distribution.
Keep the default options and click okay. In the results folder, a new histogram section will have been generated. Select the frequency distribution section, then click the dropdown insert menu and select new analysis XY non-linear regression curve fit.
Click on Gaussian distribution and click okay. In the results window that appears the mean and standard deviation are given. Generate a threshold by adding 1.96 standard deviations to the mean return to the Excel file.
Create a new tab called combined label column A accession and copy all the accession numbers from each of the individual experiments into this single column. Next, select the data tab and then the remove duplicates option. Then create columns for the SLAC ratios and ratio variability from each experiment as well as for the protein name description column.
In the description tab, use the VLOOKUP formula to collect the description of each accession number. Drag the formula down the column to fill in the protein description, and then use this formula to obtain the ratio and variability data from the individual experiments. To highlight interacting proteins in the combined dataset, note the threshold value for a particular experiment.
Select the ratio column and click on the home tab. Followed by conditional formatting, highlight cells rules more rules, then select style classic format only sells that contain sell value greater than or equal to. In the box, type in the 1.96 standard deviation value and click okay.
Assess the ratio variability for each positive interaction. If subtracting the percent variability would drop a hit below the threshold value. This should be treated with caution.
Repeat this for each of the results columns and compare across experiments highlighted proteins identified in two or more experiments represent a high confidence interaction. Western blot analysis confirmed the purification of the GFP tagged protein of interest. The lack of a gap DH band in the bound samples indicates that non interacting proteins were depleted from the samples by the immunoprecipitation step, whereas the presence of an EIF 4G band in these samples indicates known interacting binding partners were retained.
Several of the identified EIF four A binding proteins were conserved between the two isoforms. The proteins that interact with EIF four A are identified by the high LAC ratio, whereas non-specifically bound proteins in this experiment have ratios below 0.96. In this diagram of the initiation factor complex, the EIF four A binding proteins that were identified in the SLAC amino precipitation experiment are shaded in red to white.
According to the log two SLAC ratio, the high coverage of both direct and indirect binding partners of the EIF four A one and two complexes illustrates the effectiveness of this approach for identifying protein interactions While attempting this procedure, it's important to remember that while some variation in protein levels can be compensated for at the data analysis stage, it is best if this can be avoided by ensuring accurate normalization of protein input levels and by avoiding losing aros beads during the wash steps.
