How cells maintain protein homeostasis is not well understood in the context of cellular stress and senescence. In this protocol, we provide a simple and accurate way to calculate protein turnover, which is a key component of protein homeostasis in living cells. Pulse SILAC gives an experimenter the ability to measure protein turnover for individual proteins and the entire proteome in a high-throughput manner that is also more authentic to biological systems than other methods.
The principles of this method can be applied to any system that can be metabolically labeled, including whole organisms. This method can also lead to insights into cellular senescence, aging, and neurodegenerative disease. Mass-spectrometry analysis is highly sensitive to certain chemical contaminants, such as detergents.
Use LC-MS-grade reagents throughout this procedure to ensure high-quality data collection. To begin the metabolic labeling of the cells, replace the media for three plates each of senescent and quiescent cells with 30 milliliters of SILAC Light media. Separately, replace the medium for three senescent and three quiescent plates with 30 milliliters of SILAC Heavy.
Grow the cells for three days without changing the medium. To detach the cells from the culture plates, add five milliliters of pre-warmed trypsin reagent to each plate and incubate the plates for five minutes at 37 degrees Celsius. Later, resuspend the detached cells in five milliliters of the same media used for culture to a total volume of 10 milliliters.
Next, transfer the cells to ice and centrifuge the cells at 300 times G at four degrees Celsius. After washing the pellet twice, spin down the cells again and remove the supernatant before resuspending the pellet in 150 microliters of freshly prepared eight-molar urea and 50-millimolar ammonium bicarbonate lysis buffer. Mix the content of the tube by pipetting up and down.
Aliquot 50 micrograms of the protein sample into a new tube and brings samples up to equal volumes with lysis buffer. Then add dithiothreitol to a final concentration of 20-millimolar to the tube and incubate the sample at 37 degrees Celsius for 30 minutes with shaking. Allow the sample to cool at room temperature for 10 minutes before adding iodoacetamide to a final concentration of 40-millimolar and incubating at room temperature in the dark for 30 minutes.
Then dilute the sample to below one-molar urea with a 50-millimolar ammonium bicarbonate buffer. Add one microgram of trypsin for 50 micrograms of starting protein or at 1:50 trypsin to protein ratio to the sample. Incubate the sample overnight at 37 degrees Celsius with shaking to digest proteins into peptides.
The next day, add formic acid to 1%by volume of the sample to quench the protein digestion. For the sample cleanup, place a solid-phase extraction cartridge on a vacuum manifold, using one extraction cartridge for each sample, according to the manufacturer's guidelines. After the extraction, remove the peptide samples from the vacuum manifold and dry them completely in a vacuum concentrator.
Drying takes approximately three hours. For the mass spectrometry, or MS analysis, resuspend the peptide sample at a concentration of 400 nanograms per microliter in a 0.2%formic acid buffer. To resolubilize the peptides, mix the sample for five minutes.
Then, sonicate for five minutes in a water bath sonicator. Then, centrifuge the sample to pellet the insoluble materials and transfer the peptide supernatants into MS vials. Submit the samples for the proteomic analysis using liquid chromatography tandem mass spectrometry, or LC-MS-MS.
Use the settings recommended for untargeted analysis by the mass spectrometry facility. Load the samples onto an analytical column with a flow rate of 400 nanoliters per minute to elute the peptides over a 90-minute linear gradient using a mixture of organic and inorganic solvents, with the organic solvent ranging from 5 to 35%Acquire mass spectrometry data in the data-dependent mode, with a continuous cycle of MS1 survey scans followed by 20 data-dependent MS2 scans with HCD fragmentation. Quantify the peptide peak areas for heavy and light peptides in Proteome Quantitation software tool.
Then, export the heavy and light peptide peak areas. For the calculation of protein half-lives, open the SILAC Analysis Workbook to the first sheet named Raw Data and paste in the UniProt IDs, gene names, heavy and light peak areas into the indicated columns. Then, open the fourth sheet named Analysis and remove the rows where columns G and H indicate that the sample is out of range and keep rows that read within the range.
The senescent phenotypes were validated using the senescence-associated beta-galactosidase activity and RT-qPCR analysis. For the beta-galactosidase activity, the senescent cells appeared blue, while the quiescent control cells had no or minimal color. In the RT-qPCR analysis, the senescent cells displayed higher interleukin-6, CXCL8, and CDKN1A-P21 mRNAs compared to the quiescent cells.
Conversely, the level of lamin B1 encoding for proliferation marker was low or absent in the senescent cells. The extracted ion chromatograms of peptides revealed the relative proportion of heavy and light peptide signals. A lower proportion of heavy peptide signals relative to light senescent cells indicated a slower protein turnover rate, and a higher heavy peptide signal relative to light indicated a faster protein turnover rate.
The unlabeled samples showed little or no heavy peptide signal. The half-lives for 695 proteins identified from the mass spectrometry analysis were calculated. The half-life is reported as a log-two ratio of senescent over quiescent cells and plotted in a volcano plot.
Following the calculation of protein turnover rates in senescent cells, downstream analysis may reveal candidate biological pathways that are regulated by proteostasis mechanisms. Pulse SILAC paved the way for researchers to study protein dynamics with unprecedented granularity and throughput, enabling us to study changes in protein homeostasis in many individual proteins.
