Analysis of the unlinked sugar chains on glycoproteins starts with the labeling of the glycans and cells with tritiated manos. The pulse dish is cooled on ice while the chase dish is incubated with no label for a desired chase period. Next, the samples are lysed and a glycoprotein of interest is immuno purified Denatured and its glycans are enzymatically released.
The oligosaccharides are further purified by molecular filtration, which separates them from the proteins and other contaminants larger than 30 kilodaltons. The glycans are then separated by HPLC. The amount of each glycan form is determined by measuring the intensity of the radioactive signal in each eluded fraction.
Hi, I'm Edward Azo from the laboratory of Hi Geraldo Letter Kremer in the Department of Cell Research and Immunology at Tel Aviv University. I'm also from the Gerardo Letter Kremer Slab. And today we'll show you a procedure for isolating and analysis of metabolically labeled and linked oligosaccharides.
We use this procedure in our laboratory to study the dynamics of sugar chain processing for both total cellular glycoprotein pools and individual glycoproteins. Okay, so now let's get started. To begin this procedure, use NIH three T three cells grown overnight in one 90 millimeter dish.
For each sample, the culture is sub confluent to starve the cells for glucose, prepare fresh two x glucose free medium supplied with 20%dialyzed, FCS and eight millimolar Sodium pyruvate dilute one volume of the desired amount of the two X medium with one volume of deionized water. Prewarm the medium, then incubate each cell sample in five milliliters of the fresh pre-war medium for five to 15 minutes. If chase is longer than 12 hours are required, the cells should be handled in sterile conditions.
At the end of the incubation period, mark the dishes corresponding to pulse and chase samples. To label the n glycans for glycoprotein analysis by immunoprecipitation, replace the starvation medium with one milliliter of prewarm glucose free medium supplemented with 400 micro curies per milliliter of two tritium labeled manos. Use 65 micro curies per milliliter for labeling and analysis of total glycoproteins.
After replacing the starvation medium, incubate the cells in the normal tissue culture conditions for one hour. At the end of the one hour incubation, remove the labeling medium from all the samples Carefully add two milliliters of PBS at four degrees Celsius to the samples corresponding to pulse and place them on ice. These samples are referred to as pulses.
Use two milliliters of prewarm normal complete culture medium. To rinse the chase samples three times. Then add five milliliters of the pre-war regular medium to the cells.
Place the chase samples in a carbon dioxide incubator at 37 degrees Celsius for the desired chase periods. During the incubation of the chase samples, rinse the pulse samples, which were left on ice three times with two milliliters of ice cold PBS. Then use a cell scraper to scrape the cells off the dish.
In two milliliters of PBS, place the released cells into a two milliliter EOR tube.Short. Spin the cells at 16, 000 GS for six to 10 seconds to scar the supernatant and freeze the cell pellet at minus 80 degrees Celsius. When the incubation of the chase samples is over, rinse them.
Spin and store as just shown for the pulse samples. Once ready to continue to cell lysis, remove the cells from the freezer. For the IP analysis ly the cells by adding 300 microliters of buffer.
A vortex briefly and incubate for 20 minutes on ice. For analysis of the samples of total glycoproteins at 300 microliters of buffer B.Incubate the cells for 20 minutes on ice, followed by sonication for four times, 10 seconds each time at maximum amplitude. Then boil the samples for five minutes.
Finally, centrifuge all the lysates at 16, 000 Gs for 20 minutes at four degrees Celsius. Transfer the supernatant to a new EOR tube and discard the pellet. Proceed to IP for examining a protein of interest for immunoprecipitation of the H two A glycoprotein demonstrated here.
Add 20 microliters per sample of protein, A agro speeds and three microliters per sample of the lab's rabbit polyclonal anti H two A antibody to the supernatant of the lysed cells. Incubate the samples for four to 16 hours at four degrees Celsius, constantly mixing by slow rotation. At the end of the incubation, spin down the beads and carefully remove the supernatant using a vacuum, rinse the beads four times by adding 500 microliters of buffer tea.
Four texting spinning down the beads and removing the supernatant to release the bound proteins from the beads. Add 10 microliters of denaturing buffer from the endo H kit to the bead pellet and boil the samples for five minutes. Spin down the beads and transfer the supernatant into a new EOR tube.
The ellu proteins are now ready for deglycosylation. The glycans are released from both the IP and total protein samples using the enzyme and OH.For the IP samples, add 0.5 microliters of endo H enzyme to each sample along with 10 microliters of the two x endo H reaction buffer. Incubate the samples at 37 degrees Celsius for three hours.
For total glycoproteins, apply the supernatant of the cell lysates to a MicroCon filter with a 30 kilodalton cutoff. Wash the total protein extract three times with 100 microliters of buffer E by repeated centrifugation at 14, 000 Gs.For three minutes, add three microliters of 10 x endo H reaction buffer, and 1.5 microliters of the endo H enzyme to the MicroCon. Ate where the washed proteins remain and incubate for three hours at 37 degrees Celsius alongside the IP samples.
At the end of the three hours incubation, dilute the IP sample five times with deionized water and place on top of a new MicroCon filter and centrifuge at 14, 000 Gs.For three minutes. Proceed to wash out both the IP and total protein samples by applying 50 microliters of deionized water and centrifuging the samples Repeat for two more times. Keeping the flow through which contains the cleaved glycans if desired, through Tates containing endo H.Resistant glycoproteins are then incubated with 200 milli units of N glycosate F in 15 microliters of buffer C at 37 degrees Celsius for 16 hours.
Elution with deionized water is done as just shown. Dry the samples completely using a speed back concentrator with heating up to 45 degrees Celsius to accelerate the process. Re suspend the dry pellets in 12 microliters of the HPLC solvent and load the samples on a sphe or amino column installed in the HPLC device.
Run the samples as described in the accompanying written protocol. Collect 48 fractions for each sample run. Use a scintillation counter to measure the CPM values of the collected fractions and plot the CPM readout as a function of fraction number.
Calculate the percent of manos residues of all glycan species as described in the accompanying written protocol. Here are representative results of sugar chain analysis of total glycoproteins from a one hour pulse labeling sample. The CPM readout of the HPLC fractions is plotted as a function of the fraction number in the resulting profile.
In each peak represents a specific glycan species identified by an HPLC run of known glycan standards. For each glycan species, its molar percent of total glycans is plotted as a function of the chase. Time shown here is such an analysis for a specific misfolded glycoprotein isolated by immunoprecipitation from a sample treated with the proteasome inhibitor MG 1 32.
The trimmed glycan species M five six accumulates during the chase period leading to the conclusion that oligosaccharides found on a misfolded undergo extensive man trimming prior to degradation. This is in contrast to oligosaccharides on total glycoproteins from samples treated in the same way Here, M nine eight remain the major glycan species during the chase with only a minor increase in the amount of M five six. This leads to the conclusion that only a small fraction of the total glycoprotein pool is targeted to ER associated protein degradation.
Abbreviated rad. As explained further in the accompanying written protocol, We've just shown you how to use a combination of palase in live cells and HPLC analysis in order to follow the dynamics of unlinked oligosaccharide composition during the life of glycoprotein. When doing this procedure, it's important to remember to avoid contaminations of chase samples with traces of a radioactive manners, which will result in the unwanted labeling of the glycans during the chase periods During the size separation.
It is also important to adjust the HPLC to a constant pressure in order to ensure the consistency of the fractionation of the glycans. So that's it. Thanks for watching and good luck with your experiments.
