This protocol is an easy way to generate the profiles of ubiquitin and ubiquitin-like post-translational modifications by identifying and semi-quantifying specific identified proteins. The main advantage of this technique is the use of lentiviruses to create stable cell line of expression within a week, and the use of two tags to specifically purify proteins. In fact, this kind of post-translational modifications are involved in most biological functions, and alterations of this mechanism are found in most disease.
Therefore, identifying these alterations may serve as a prognostic and/or diagnosis and of course as molecular targets. Ubiquitin is one of the most conserved protein in eukaryotic cells and this essential for their survival. Therefore, this kind of method could be applied to any eukaryotic model, from mammals to yeast.
However, our lentiviral system is restricted to the study of cells. Since the whole procedure is quite long with sometimes long incubation time, it is possible to do it over two days instead of one. The eluate from nickel beads can be frozen at minus 20 and before adding the Flag beads.
This protocol contains many steps among which some are critical in order to guarantee the final success of the purification, and so the accurate establishment of the post-translational modification profile. Demonstrating of the procedure will be performed by Mirna Swayden and Aurelie Dobric, two PhD students of the lab. To begin, add 0.5 times 10 to the sixth of HEK 293T cells in a six-well plate with two milliliters per well of DMEM with 10%FBS to seed, and incubate at 37 degrees Celsius, 5%carbon dioxide, and 100%humidity.
The next day, 50 to 70%confluence is achieved. Cotransfect the cells with a mix of one microgram of pCCL-6HF ubiquitin-like or pCCL-GFP, one microgram of PBS VG, and one microgram of delta helper vectors using a transfection reagent and protocol for lentivirus production. After six hours of transfection, change the medium to a fresh one corresponding to the media of cells to be transduced.
Seed the cells to be transduced in a six-well plate with their standard culture media in order to obtain 10 to 20%confluence the day after. 24 hours after transfection, recover the medium containing lentiviral particles, and filter using 0.45-micron filters. Replace the medium of the cells to be transduced by the filtered medium containing lentiviruses.
Incubate the cells with lentiviruses for 24 to 72 hours in an incubator at 37 degrees Celsius and 5%carbon dioxide, and then change the medium with fresh, standard one. Check GFP expression using an inverted fluorescent microscope to evaluate efficiency of transduction. If no fluorescence is detected, wait for an additional two to three days.
If the GFP control is positive with green fluorescence, grow all cells until having enough to perform an expression control of 6-His-FLAG ubiquitin-like by immunofluorescence, as shown here, and western blot using anti-FLAG antibody. After growing the cells in 15-centimeter dishes, wash the culture dishes at least one time with phosphate-buffered saline at room temperature. To begin cell lysis, add two milliliters of buffer one into each 15-centimeter dish at room temperature.
Use a cell scraper to recover all lysates into 50-milliliter conical centrifuge tubes. Sonicate the lystates three times for 30 seconds, separated by a one-minute pause. Centrifuge the sonicated lysates at 15, 000 times g for 15 minutes.
Transfer the supernatant to a new tube through a 40-micron cell strainer. It's very important to filter lysates after sonication and centrifugation. Not doing this may result in the copurification of many non-specific proteins, thereby increasing the background and strongly reducing the size of the PTM profile.
Transfer an equal amount of protein between 50 and 100 milligrams to new Falcon tubes. Add nickel ion NTA beads into each tube with the amount of two microliters of beads per one milligram of protein. Place the tubes on a rotator to rotate at 30 rpm for 2 1/2 hours at room temperature.
Then pellet the beads at 500 times g for five minutes. Wash the beads with one milliliter of buffer one, and transfer the samples to a 1.5-milliliter microcentrifuge tube. Place the tube on ice.
Wash twice with one milliliter of ice-cold buffer two containing ten-millimolar imidazole. To elute-bound proteins, add 600 microliters of buffer two containing 250-millimolar imidazole, and rotate for two hours at four degrees Celsius. After pelleting the beads by centrifugation at 500 times g for one minute, transfer the supernatant to a new pre-cooled 1.5-milliliter tube, and add 50 microliters of anti-FLAG M2 antibody-conjugated beads.
Rotate at 30 rpm for 2 1/2 hours at four degrees Celsius. Then wash twice with 500 microliters of buffer two, then twice with 500 microliters of buffer three. For the final elution, add 100 microliters of buffer three containing a FLAG peptide at 0.01 microgram per microliter, and rotate at four degrees Celsius for 1 1/2 hours.
After centrifugation at 500 times g for one minute, transfer the supernatant to a new pre-cooled tube. Take 10 microliters to load on SDS-PAGE, and perform a silver staining of the gel to control the purification quality. If the purification looks good, analyze the 90%left by LC-MS.
To perform normalization, use the formulas to normalize values between drug-treated cells and untreated cells for ubiquitin and GFP. To remove background, subtract values in the control sample GFP from values in the ubiquitin samples, to obtain specific values for each identified protein in both conditions. To obtain the variation of ubiquitination, obtain a score between minus 100 and plus 100 for positive and negative variations of PTMs induced by a drug.
The difference between the specific values of treated and untreated samples are divided by the sum of all values, including those in the control, and multiplied by 100. Variations below minus 50, representing repression of PTM, or above 50, representing induction of PTM, are considered as significant. Use this formula to obtain a confidence value between zero and 100%Values above 50 are usually considered to be confident.
To obtain a nicer distribution of induction and repression values, and to consider both variation and confidence parameters, use this formula to multiply the variance and confidence values. In this study, transduction of culture mammalian cells was achieved to create GFP and 6-histidine FLAG ubiquitin expressing cells. The efficacy of lentiviral transduction was first controlled by looking at the GFP fluorescence of GFP-transduced cells.
The expression of FLAG-ubiquitin was done by immunofluorescence staining using an anti-FLAG antibody, which shows the percentage of transduced cells. In order to control the expression level of exogenous FLAG-ubiquitin, lysates from transduced cells were analyzed by SDS-PAGE followed by western blot with anti-FLAG antibody. After purification of the ubiquitinated proteins, 10%of the final elution was used to control the amount and integrity of purified material by SDS-PAGE and silver staining of the gel.
Background GFP sample subtraction identified 364 proteins significantly ubiquitinated with a score above 50%A gene set enrichment analysis of these ubiquitinated proteins was performed in order to highlight the biological processes in which they were involved. Potential interacting networks formed by gemcitabine-induced alterations of ubiquitination. This led to the identification of functional interacting networks strongly affected by increased or decreased ubiquitination of the involved proteins.
It was also confirmed that ubiquitination of PCNA increased after gemcitabine treatment. It's very important to avoid the transfer of some beads after both elution steps, since it may result in a significant increase of background. This procedure will generate specific post-translational modifications which, by comparing different conditions, will enable the identification of specific alterations.
The first next step will be validate at least the alterations of interest based on biochemical approaches. We developed this method to explore ubiquitin-based, post-translational modifications in order to identify new ways and mechanisms of pancreatic cancer cells. Since this protocol and other developed worldwide have been employed successfully to decipher different biological processes.
The lentiviruses have to be used at least in BL-2 laboratory. Guanidine is a strong denaturing agent and has to be used carefully. The sonicator can also be harmful for ears and have to be used following recommendations.
