The oligo mass profiling or Olympic experiment shown here allows one to gain rapid insights into the structure of polymers in the extracellular matrix or cell wall of an organism. In the case here, cell walls are prepared from plant seedlings by first removing cellular components with 70%ethanol. The target wall polymer is next digested with a specific hydro lace solubilizing particular wall components in the form of all ligaments.
An alternative approach is to digest the cell wall with the enzyme in C two directly on the tissue. The relative abundance of the release de ligaments is determined by MALDI to mass spectrometry and statistical analysis. Hi, I'm Marcus Polly from the Energy Biosciences Institute here at the University of California in Berkeley.
Hi, I'm Marcus Re.And I'm Asha Giller. Also from the Energy Biosciences Institute. Organisms are surrounded by extracellular matrices that depending on the organism consist of complex polymer networks.
A rapid and sensitive way to assess the extracellular matrix is a method we call oli mass profiling short oleum, which we will show you today. This procedure can also be performed directly on the plant tissue. So let's get started.
Begin this procedure by isolating the extracellular matrix or cell wall of plant tissues. Harvest five arabidopsis seedlings or the equivalent amount of other plant material and transfer them into a 1.5 milliliter reaction tube. Make sure that the material is placed at the bottom of the tube.
Add two three millimeter metal balls to the reaction tube and snap freeze in liquid nitrogen. Then grind the frozen sample using a ball mill for 2.5 minutes at 25 hertz. After grinding the material, add one milliliter of 70%aqueous ethanol to the sample.
Remove the metal balls using a magnet and vortex thoroughly. This treatment solubilize most proteins and cell organelles centrifuge the sample at 14, 000 RPM for 10 minutes to pellet the alcohol insoluble residue. Representing the cell wall material carefully aspirate the supernatant, making sure not to disturb the pellet.
Add one milliliter of chloroform methanol solution to the pellet remaining in the tube vortex thoroughly. To resuspend the pellet and centrifuge the chloroform methanol extracts hydrophobic compounds such as lipids. After centrifugation, remove the snat and dry the pellet completely Using a vacuum centrifuge, this completes the isolation of the cell wall material, which can now be treated yield specific ligaments.
The example demonstrated here is the solubilization of Xlo glucan, the major hemi cellulose present in the cell walls of dicot plants such as Arabidopsis Xlo glucan is a polymer consisting of a glucan backbone that is heavily substituted with other sugar residues for the solubilization of this polymer. A fungal endo glucan is used to solubilize this hemi cellulose Resus suspend the dry isolated cell wall pellet in 25 microliters of 50 millimolar ammonium formate pH 4.5 and add 0.2 units of endo gluconate. Vortex the suspension and spin the liquid down.
Place the tube in a mixer and let the walls digest for 16 hours at 37 degrees Celsius with shaking. At 300 RPM. When digestion of the isolated cell wall is complete, remove the solvent using a vacuum centrifuge and proceed to analyze the released oligosaccharides by mass Spectrometry, salts and enzyme are removed from the digested sample using the cation exchange resin beads from Biox MSD 5 0 1.
The beads are first conditioned by placing the needed amount in an empty column and washing the beads with copious amounts of water. Add five to 10 conditioned biox cation beads to the digested and dried sample. Then add 10 microliters of water for lower amounts of material.
Five microliters of water may be used. Immerse the beads in the solution by briefly spinning the tube incubate for at least 10 minutes at room temperature while the digest is incubating with the beads. Spot two microliters of DHB matrix onto a mal D target plate.
Evaporate the matrix solvent under vacuum leading to matrix chemical crystals now spot two microliters of the Desalted Digest solution. On top of the matrix crystals on the target plate. The solvent of the sample matrix spot is again evaporated under vacuum.
It is important to maintain an appropriate length of time for mixing of the sample matrix liquid prior to crystallization as described in detail in the accompanying written protocol. Once the sample has dried, place the target plate into a maldi to mass spectrometer. Set the machine to positive mode with an accelerating voltage of 20, 000 volts and a delay of 350 nanometers.
The selected mass range for these hemi cellulose ligaments is 500 to 3000 Daltons. The mass range should be modified according to the size of the expected ligaments. Start firing the laser and collect 100 to 200 spectra per sample, which are compiled to generate a representative average spectrum.
This is the Hemi cellulose oly spectrum. The oly method demonstrated in this video can also be used directly on the plant tissue, omitting any wall preparation steps. This in situ analysis procedure is demonstrated on dark grown arabidopsis seedlings.
The specimen is placed directly onto a target plate and air dried to the dry seedling on the plate. Add 0.5 microliters of the appropriate enzyme buffer mix at the desired positions. Make sure that the enzyme drop touches partly the tissue and the target plate.
Place the maldi target plate in a closed container with saturating humidity to avoid drying out of the enzyme drops. Incubate the plate for 16 hours at 37 degrees Celsius. At the end of the incubation period, dry the enzyme spot under vacuum and add 0.5 microliters of the liquid DHB matrix on top of each dried enzyme spot.
Then dry the MALDI target plate under vacuum. To analyze the NC two samples. Place the target plate, including the tissue specimen enzyme matrix spots into a multi to mass spectrometer record mass spectra, using the same settings as for the isolated cell wall sample.
Start firing the laser onto the matrix. Sample crystals next to the tissue. Make sure not to hit the tissue itself as in such a case, the time of flight of the molecules will be off.
Collect around 20 to 50 spectra per spot and compile them to generate a representative Average spectrum. This is a representative average spectrum of the oligosaccharides derived from the HemosIL glucan present in AOPs. The seedlings, the ion intensities, or ion height of all the ions of interest are added up to 100%resulting in the relative abundance of the various oligosaccharides.
The result of the NC two OMP method gives similar results. Each enzyme digestion droplet marked by a colored circle can be analyzed independently and the corresponding mass spectra can be obtained and analyzed. Oola can also be performed on other polymers of the extracellular matrix if an appropriate hydrolyzing enzyme is available.
As an example, this is an oola spectrum of the hemo cellulose cylin present in the bioenergy crop. Ms.Canus to obtain the presented data while material from a mecampus leaf was digested with the xin. For the assignment of structures to the ions, please refer to the accompanying protocol.
We've just shown you the procedure of oligo mar mass profiling on exo cellular metrics. Polymers, When doing this procedure, it's important to remember that best results can be obtained when homogeneous matrix results are obtained for the OV MS analysis. So that's it.
Thanks for watching and good luck with the experiments.
